in vitro and in vivo models of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 408253 10 pageshttpdxdoiorg1011552013408253

Research ArticleEndothelial Gene Expression and Molecular Changes inResponse to Radiosurgery in In Vitro and In Vivo Models ofCerebral Arteriovenous Malformations

Jian Tu1 Zhiqiang Hu2 and Zhongbin Chen3

1 Australian School of Advanced Medicine Macquarie University 2 Technology Place North Ryde Sydney NSW 2109 Australia2 Department of Neurosurgery The 9th Medical Clinical College of Beijing University Beijing 100850 China3Department of Electromagnetic and Laser Biology Beijing Institute of Radiation Medicine Beijing 100038 China

Correspondence should be addressed to Jian Tu jamestumqeduau

Received 10 May 2013 Accepted 31 August 2013

Academic Editor Saulius Butenas

Copyright copy 2013 Jian Tu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Radiosurgery for cerebral arteriovenous malformations (AVMs) is limited to 2-year latency There is no early marker to monitorwhether the lesion is responsive to radiosurgery In this study we examined endothelial gene expression and molecular changes inresponse to radiosurgery Gene expression of E- and P-selectin ICAM-1 PECAM-1 VCAM-1 tissue factor and vWF in humancerebral microvascular endothelial cells was quantified by RT-qPCR at different radiation doses and time points Soluble E- andP-selectin ICAM-1 VCAM-1 and tissue factor in an animal model of AVMs were quantified by ELISA at different time afterradiosurgery We found that gene expression of E- and P-selectin ICAM-1 PECAM-1 and VCAM-1 was upregulated by radiationin a dose-dependent manner (119875 lt 05) Gene expression of E- and P-selectin and ICAM-1 was more sensitive to irradiation thanthat of PECAM-1 andVCAM-1 Radiosurgery induced gene expression of P-selectin ICAM-1 PECAM-1 andVCAM-1 was linearlycorrelated with time (119875 lt 05) Radiosurgery induced elevation of soluble E- and P-selectin ICAM-1 VCAM-1 and tissue factorin a rat model of AVMs (119875 lt 05) Thus a combination of these molecules measured at different time points may serve as an earlypredictor of responsiveness of AVMs to radiosurgery

1 Introduction

Cerebral arteriovenous malformations (AVMs) consist oftangles of immature vessels which are one of the majorcauses of hemorrhagic stroke in children and young adultsCurrent treatment options include surgery endovascularembolization and radiosurgery Surgery is only performedwhen the operative risk is lower than the morbidity andmortality associated with the natural history of the lesionEmbolization is mainly applied to reduce the size of theAVM nidus prior to definitive surgical or radiosurgicalmanagement Radiosurgery offers a valuable addition to thetreatment armamentarium for those patients who are eitherunable to undergo surgical resection or unable to be accessedwith endovascular catheters for endovascular embolizationof AVMs Radiosurgery induces vasoocclusion a processthat involves a combination of cellular proliferation and

intravascular thrombosis It takes 24 years on average toocclude all vessels within the lesion [1] Partial vasoocclusiondoes not reduce the risk of AVM rupture [2]

Vasoocclusion can occur as early as 4 months or as lateas 5 years after radiosurgery or may never occur by the endof the follow-up period [3ndash6] To date angiography is thegold-standard for confirming obliteration and is performed2-3 years after radiotherapy [5] Early partial response toradiosurgery is difficult to be angiographically assessed in areliable manner An early marker that reflects AVM vesselresponses to irradiation is needed to predict the effectivenessof radiosurgery

Following radiosurgery AVM endothelial cells undergoapoptosis release IL-1120573 which acts as an autocrine signal toenhance endothelial cell apoptosis and also as a paracrinesignal to induce expression of endothelial adhesionmoleculesand proinflammatory cytokines on surrounding endothelial

2 BioMed Research International

cells [7] Expression of adhesion molecules including E-selectin P-selectin ICAM-1 PECAM-1 and VCAM-1 isupregulated following irradiation [8] E-selectin is exclusivelyexpressed on the surface of activated endothelial cells [8]and is a critical mediator of leukocyte rollingmdashthe first stepin the cascade of events that lead to firm adhesion andtransmigration into the inflamed tissues [9] The mechanismof induction of E-selectin expression is likely to be via acti-vation of NF120581B by reactive oxygen intermediates generatedby irradiation [10] P-selectin is stored in the Weibel-Paladebodies of endothelial cells Like E-selectin P-selectin is acentral mediator of leukocyte rolling during inflammationand is also able to mediate the adherence of platelets toneutrophils and monocytes [11] allowing this molecule toparticipate in the cross-talk between the inflammatory andthrombogenic processes In addition to its role during inflam-mation P-selectin also participates in coagulation by bindingwith tissue factor to accelerate the formation and depositionof fibrin during thrombogenesis [11] ICAM-1 is constitu-tively expressed on the cell surface of endothelial cells andleukocytes and functionally activates leukocyte-endothelialadhesion and migration [12] The mechanism of inductionof ICAM-1 appears to be similar to that of E-selectin withactivation of NF120581B upregulating transcription of the ICAM-1 gene [13] The expression of PECAM-1 is restricted to cellsof the vascular system The molecule is highly expressed atthe lateral junctions of endothelial cells and on the surfaceof platelets and leukocytes [14] PECAM-1 is required inthe initial formation of endothelial cell-cell contacts [15]and involved in thrombosis [15] VCAM-1 is constitutivelyexpressed on endothelial cells [16] andmediates tethering androlling of lymphocytes [17] and monocytes [18]

Similar to the process of endothelial apoptosis leading toaltered inflammatory properties the coagulative propertiesare also upregulated via increased expression of thromboticmolecules including von Willebrand factor (vWF) [19] andtissue factor [20] vWF is synthesized in endothelial cells andstored in Weibel-Palade bodies Radiation increases consti-tutive synthesis of vWF and exocytosis of the Weibel-Paladebodies Endothelial secretion is the principal source of vWFin the plasma vWF plays a critical role in platelet adhesionand coagulation as well as being important in leukocyterecruitment during inflammation [21] Circulating vWF actsas a carrier protein for factor VIII and prolongs the half-lifeof factor VIII in the circulation by inhibiting the inactivationof factor VIII by factor Xa [22] vWF that is released duringthrombus formation prevents rapid clearance of factor VIIIfrom the plasma thereby promoting thrombin generation[23] Tissue factor is the primary initiator of the extrinsiccoagulation cascade and it is not expressed on endothelialcells but present on subendothelial cells and in the vascularadventitia Under normal conditions tissue factor is anatom-ically sequestered from the blood In response to endothelialdamage or cytokine stimulation tissue factor is activated [24]and forms a complex with factor VIIaThe tissue factor-factorVIIa complex catalyses the activation of factor IX and factorX leading to the conversion of prothrombin to thrombin andthence to the production of fibrin from fibrinogen [22 24]Both blood-borne tissue factor and tissue factor present in the

vessel wall contribute to the initiation and propagation of theprocesses Tissue factor-rich microparticles circulate withinthe blood and serve as a major source of blood-borne tissuefactor [11]

Based on our previous work that endothelial molecularchanges observed in human cerebral AVMs an animalmodelof AVMs and a murine endothelial cell model in responseto radiosurgery [25ndash31] we hypothesize that expression ofendothelial molecules could be upregulated at transcriptionallevel and detectable in blood after radiosurgery The objec-tives of this study were to test our hypothesis in a humancerebral microvascular endothelial cell model as no biopsyhuman AVM specimens that well responded to radiosurgerytreatment are possibly obtained and an AVM animal modelto establish a time course of endothelial molecule changesand identify early predictors of the ultimate response toradiosurgery

2 Materials and Methods

21 Chemicals Primers Reagents andCell Culture All chem-icals M199 and RPMI1640 phenol red free media and cellculture supplements were purchased from Sigma-Aldrich (StLouisMOUSA) unless otherwise specifiedAll primersweresynthesized by Sigma-Aldrich Fetal calf serum (FCS) andSuperscript kit were purchased from Invitrogen (CarlsbadCA USA) Endothelium growth factor and MTT assay kitwere purchased from Roche Applied Science (Indianapo-lis IN USA) RNeasy total RNA extraction mini kit waspurchased from QIAGEN (Hilden Germany) SYBR greenreactionmaster mix was purchased fromApplied Biosystems(Darmstadt Germany) Rat E- and P-selectins ICAM-1PECAM-1 VCAM-1 vonWillebrand factor and tissue factorELISA kits were purchased from Uscn Life Science andTechnology (Wuhan China) Human cerebral microvascularendothelial cells were purchased from Applied BiologicalMaterials (Richmond BC Canada) and maintained in M199medium supplemented with endothelium growth factor and10 FCS in a humidified atmosphere of 37∘C and 5 CO

2

Anti-rat caspase-3 antibody was purchased from Abcam(Cambridge UK) Alexa Fluor 488 conjugated anti-rabbitsecondary antibody was purchased from Molecular Probes(Eugene OR USA)

22 Cell Viability Assay following Irradiation To evaluateradiation effect on cell viability and select irradiation dosefor the following experiments human cerebral microvascularendothelialcells were examined by MTT assay followingirradiation as previously reported [27 32] Briefly humancerebral microvascular endothelial cells were seeded in 96well plates coated with haemaccel (Boehringer PharmaAmsterdam Netherlands) at a density of 8 times 103 for 48 hoursWhen cells were 60 confluent the cellmediumwas replacedby RPMI1640 phenol red free medium supplemented with10 FCS and endothelium growth factor The cell cultureswere then exposed to radiation delivered by an X-Knife linearaccelerator (Radionics Burlington MA USA) with a dose of

BioMed Research International 3

5 15 or 25Gray Sham controls were treated identically butwere not exposed to radiation

MTT was used to determine an index of cellular viabilityand mitochondrial metabolic activity MTT was prepared at5mgmL in RPMI1640 phenol red free medium The assayactivity was determined at 6 12 24 72 and 120 hours afterirradiation and performed in triplicate and repeated twice Ateach of these time points 10 120583L of MTT was added to eachwell and incubated for 4 hours at 37∘C One hundred 120583L offormanzan lysis was then added to each well and incubatedfor another 4 hours at 37∘CThe absorbance of the solubilizedformanzanwasmeasured at 570 nmusing amicroplate reader(iMark Bio-Rad Hercules CA USA)The viability of cells inirradiation sham controls was considered as 100

23 Quantification of Gene Expression following IrradiationWhen human cerebral microvascular endothelial cells were60 confluent the cells were irradiated by the X-Knife linearaccelerator (Radionics) with a dose of 5 15 or 25Gray Shamcontrols were treated identically but did not receive radiationTotal cellular RNA was isolated from the cells using RNeasytotal RNA extraction mini kit (QIAGEN Hilden Germany)according to the manufacturersquos protocol The isolated RNAwas reverse-transcribed to cDNA using the Superscript kit(Invitrogen) following the manufacturerrsquos instructions Stan-dard real-time PCR analysis of cDNA was performed byusing SYBR green reaction master mix (Applied Biosystems)and the primers listed in Table 1 at 60∘C to 95∘C for 45cycles in the Sequence Detection System (ABI Prism 7000Applied Biosystems) following the manufacturerrsquos instruc-tions The amount of PCR products was normalized usinga housekeeping gene (120573-actin or GAPDH) to calculate therelative expression ratios Each experiment was performed intriplicate and repeated at least twice Values were comparedwith those of the control-RNAobtained from sham irradiatedcells The fold-changes of the expression were calculatedusing threshold cycle (Ct) values

24 AVM Animal Radiosurgery Animal experimentationwas approved and performed in accordance with the guide-lines of the corresponding institutional Experimental AnimalCare and Ethics Committee and the Code of Practice for theCare and Use of Animals for Scientific Purposes [33] AVMrat model was surgically created by an anastomosis of theleft common carotid artery to the left external jugular veinas detailed in our previous reports [25 26 28 34]

Sprague-Dawley male rats (7 weeks old 230 plusmn 9 g) werehoused in a parasite-free environment Standard rat cageswere used to house three rats per cage and bedding waschanged twice a week Food and water were provided adlibitum Ratswere allowed to acclimatize to new surroundingsbefore the experiment began Surgical procedures were notperformed in the presence of other rats General anaesthesiawas induced using a mixture of 4 isoflurane and oxygen(2 Lmin) via a nose cone The depth of anaesthesia wasassessed using the respiratory rate and by checking the hindlimbwithdrawal to pain No procedure was commenced until

there was a consistent absence of response to pain A heatingblanket was used for the duration of the procedure

The procedure was performed in a sterile field usingaseptic technique The left common carotid artery (CCA)was exposed and blood flow was measured through theCCA using a 1mm Doppler ultrasonic probe (TransonicSystems Ithaca NY USA) The left external jugular vein(EJV) was then exposed and ligated with 100 nylon suture atits junction with the subclavian vein An aneurysm clip wasplaced across the rostral EJV Microclips were also appliedproximally and distally on the CCA and a small arteriotomymade on the lateral aspect An end-to-side anastomosis ofthe EJV to the CCA was performed using a continuous100 nylon suture The clips were sequentially removed fromthe EJV distal CCA and proximal CCA Blood flow wasmeasured through the proximal CCA and the vein using1 and 2mm Doppler ultrasonic probes (Transonic SystemsInc Ithaca NY) The wound was closed and isofluranewas turned off allowing the animal to breathe oxygen untilthe time of awakening Once awake the animal was placedin an individual cage and housed singly for one weekpostoperatively Observations were carried out daily for thefirst week and then weekly thereafter Observations includedweight assessment of motor function behavior and woundhealth

Six weeks after surgery radiosurgery was performedunder intramuscular ketamine and midazolam anaesthesiaThe depth of anaesthesia was assessed using the respiratoryrate and by checking the hind limb withdrawal to pain Oncesedated the animal was placed on the stage attached tothe head ring of the X-Knife linear accelerator (Radionics)Correct positioning of the animal relative to the plannedtreatment location was confirmed by ensuring that the fistulawas placed at the intersection of the three targeting laserbeams that designate the centre of the radiation delivery arcsThe same treatment plan was used for all animals giving amaximal dose of 25Gy to the ldquonidusrdquo in the subcranial regionThe sham radiation controls were treated identically but didnot receive radiation

25 Immunohistochemistry At the completion of the exper-iment animals were anaesthetised and perfused with 4paraformaldehyde Vascular tissue from the neck regionbilaterally was harvested including proximal carotid arterycarotid-jugular anastomosis distal carotid artery arterializedfeeding vein AVM nidus and draining vein Specimens wereembedded in tissue freezing medium (ProSciTech QLDAustralia) with liquid nitrogen for immunohistochemistrySections were stained immunohistochemically as previouslydescribed [25 26 28ndash30] Briefly sections were washed inPBS at 37∘C to remove tissue freezing medium Nonspecificbinding was blocked by 10 horse serum Anti-rat caspase-3antibody was applied and incubated at 4∘C overnight Slideswere washed and incubated with Alexa Fluor 488 conjugatedanti-rabbit secondary antibody for 2 hours in dark andcoverslipped and examined using a confocal microscope(Leica SP5 Germany) and imaging data analyzed using LeicaLAS AF software Fluorescence intensity units (FIU) were


Table 1 Sequences of human gene-specific primers used for quantitative RT-PCR analysis

Primers Forward 51015840rarr 31015840 Reverse 51015840rarr 31015840

E-selectin CATGGCTCAGCTCAACTT GCAGCTCATGTTCATCTTP-selectin CAGTGGCTTCTACAACAGGC TGGGTCATATGCAGCGTTAICAM-1 GCAGACAGTGACCATCTACAGCTT CTTCTGAGACCTCTGGCTTCGTPECAM-1 CCAGTGTCCCCAGAAGCAAA TGATAACCACTGCAATAAGTCCTTTCVCAM-1 GGCAGGCTGTAAAAGAATTGCA GTCATGGTCACAGAGCCACCTTTissue factor CCCAGGCAGTCAGATCATCTTCT GCACCCAATTTCCTTCCATTTvWF TAAGAGGGCAACACAAACG ATCTTCACCTGCCCACTCCGAPDH AGCTGAACGGGAAGCTCACTGG GGAGTGGGTGTCGCTGTGAAGTC120573-actin CTGGCACCCAGCACAATG CCGATCCACACGGAGTACTTG

corrected using primary antibody controlsThe FIU has beenquantified as mean gray value

26 ELISA The assays were performed according to manu-facturesrsquo instruction Serum or plasma samples were diluted10 to 20-fold and incubated on the ELISA plate pre-coated with specific rabbit anti-rat antibody After washingthe peroxidase-labelled anti-rabbit antibodies were addedFinally the activity of bound enzyme in all kits wasdetermined with tetramethylbenzidinehydrogen peroxidesubstrate Absorbance was measured with an automatedmicroplate reader (iMark Bio-Rad Laboratories) at 450 nmSubtraction readings at 570 nm reference wavelength fromthe readings at 450 nm correct for optical imperfections inthe plate The duplicate readings were averaged for eachstandard and sample subtract the average zero standardoptical density A standard curve was generated from eachassay using the Microplate Manager 6 software (Bio-RadLaboratories) The sample concentrations read from thestandard curve was multiplied by the dilution factor Theintraassay and inter-assay precisionswere carefully controlledwithin 5 and 7 respectively

27 Data Analysis Data are expressed as means plusmn SE(number of experiments) The distribution of the studiedparameters between irradiated and sham-irradiated animalswas evaluated using Pearsonrsquos chi-square (1205942) test for ELISAresults Statistical difference between groups was determinedusing the unpaired two-tailed t-test When there were morethan two groups differences were analyzed using analysis ofvariance if the variances were equal and the Mann-Whitneynonparametric test if variances were unequal [35] Linearregressions were calculated using a statistical computer pack-age Number Cruncher Statistical Systems [35] A value of119875 lt 05 was considered statistically significant

3 Results

31 Cell Viability Radiation effect on the cell viability ofhuman cerebral microvascular endothelial cells was assessedas shown in Figure 1 The cell viability of sham irradiationcontrols was considered as 100 At 6-hour post-irradiationa range of radiation dosages from 0 to 15Gray had nosignificant effect on the cell viability The cell viability of

0

50

100

Cel

l via

bilit

y (

)

After radiosurgery (hours)

0 Gy5 Gy

15 Gy25 Gy

6 12 24 72 120

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

Figure 1 Effect of radiosurgery on cell viability Mitochondrialmetabolic activity of human cerebral microvascular endothelial cellswas determined by MTT assay as an indicator of cell viabilityfollowing radiosurgery at 0 5 15 or 25Gray at 6 12 24 72 and 120hours lowast119875 lt 05 versus 0Gray by paired comparison at the sametime point

human cerebral microvascular endothelial cells declined by28plusmn2 at 25Gray 6 hours after irradiation (119875 lt 05) At 12 or24 hours after irradiation there was no significant effect onthe cell viability at a dose of 5Gray The cell viability reducedby 25 plusmn 2 and 35 plusmn 2 at 15 and 25Gray (119875 lt 05) 12hours after irradiation respectively Further decline in the cellviability was observed at 24 hours after irradiation being 28plusmn2 and 41 plusmn 3 at 15 and 25Gray (119875 lt 05) respectively At72 hours after irradiation there was a significant reduction inthe cell viability at 5 15 or 25GrayThe cell viability recoveredby 13ndash15 in groups that received 5 15 or 25Gray at 120-hour post-irradiation The level of cell viability in the groupthat received 25Gray remained significantly lower than thatof sham irradiation controls (119875 lt 05)

32 Apoptosis of AVM Vessels In Vivo Caspase-3 is a keyenzyme in apoptotic signaling pathway and is selected asa marker for apoptosis The levels of caspase-3 expression


0

100

200

300

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43

Inte

nsity

()

After radiosurgery (days)minus2

Caspase-3

r = 06447 P lt 004

Figure 2 Apoptosis of AVM vessels induced by radiosurgeryCaspase-3 was examined immunohistochemically in AVM ves-sels before and after radiosurgery at 25Gray The intensity ofimmunofluorescence of AVM vessels was quantified using a con-focal microscope The intensity of immunofluorescence of AVMvessels observed before irradiation was arbitrarily assigned as100 Data were expressed as means plusmn SE of 4 rats at each timepoint There was a positive correlation between the intensity ofimmunofluorescence ofAVMvessels and time (119903 = 06447119875 lt 04)suggesting that apoptosis of AVM vessels was increasing with timeover a period of 42 days after radiosurgery

in an animal model of AVMs over a period of 42 daysafter radiosurgery at 25Gray were shown in Figure 2 Therewas a significant upregulation of caspase-3 expression inAVM vessels after irradiation The levels of overexpressionof caspase-3 were 72 at 1 day after irradiation and peakedat 154 at 21 days after radiation There was a positivecorrelation between caspase-3 levels and time over a periodof 42 days after radiosurgery (119903 = 06447 119875 lt 04)

33 Radiosurgery Induced Gene Expression In Vitro Radio-surgery induced endothelial gene expression was dose-responsive as shown in Figure 3 At 5Gray the relative geneexpression of E- and P-selectin and ICAM-1 was significantlyincreased by 58 plusmn 4 38 plusmn 3 and 74 plusmn 3 (119875 lt 05)respectively At 15Gray the relative gene expression of E- andP-selectin and ICAM-1 was significantly increased by 97 plusmn7 76 plusmn 6 and 113 plusmn 7 (119875 lt 01) respectively At 25Graythe relative gene expression of E- and P-selectin ICAM-1PECAM-1 and VCAM-1 was significantly increased by 126 plusmn9 141 plusmn 8 151 plusmn 8 80 plusmn 5 and 84 plusmn 5 (119875 lt 05)respectively There was no change in vWF gene expressionbefore and after irradiation Twenty-five Gray was selectedfor experiments to determine the time course of radiationinduced endothelial gene expression

Radiosurgery induced E- and P-selectin gene expressionwas shown in Figure 4 Radiation induced E-selectin geneexpression was increased more than 1-fold over 120 hoursand peaked at 48 hours after irradiation Radiation inducedP-selectin gene expressionwas increased by 15-fold at 120 hours

0

100

200

300

Relat

ive e

xpre

ssio

n (

)

Radiation dose (Gray)

E-selectinP-selectinICAM-1

PECAM-1VCAM-1vWF

0 5 15 25

lowast

lowast

lowast

lowastlowast

lowastlowastlowastlowast

lowast lowast

Figure 3 Radiosurgery induced dose-responsive endothelial geneexpression in human cerebral microvascular endothelial cellsEndothelial gene expression at 0Gray irradiation was arbitrarilyassigned as 100 Relative gene expression of E- and P-selectinand ICAM-1 was upregulated at 5 15 or 25Gray Relative geneexpression of PECAM-1 and VCAM-1 was upregulated at 25Graylowast

119875 lt 05 versus the same gene at 0Gray

after radiosurgery and the levels were positively correlatedwith time over 120 hours (119903 = 0855 119875 lt 02) Radiosurgeryinduced gene expression of ICAM-1 and VCAM-1 was shownin Figure 5 ICAM-1 gene expression was increased by 17-fold at 120 hours after irradiation and a positive correlationwas observed between gene expression levels and time (119903 =07282119875 lt 04) VCAM-1 gene expression peaked at 72 hoursafter radiosurgery and was positively increased with time(119903 = 08248 119875 lt 03) Radiosurgery induced gene expressionof PECAM-1 and vWF was shown in Figure 6 PECAM-1gene expression was doubled at 120 hours after irradiationand a positive correlation between PECAM-1 gene expressionand time was observed (119903 = 0952 119875 lt 01) No radiationresponsive vWF gene expression was observed in humancerebral microvascular endothelial cells

34 Radiosurgery Induced Soluble Molecule Changes InVivo Radiosurgery inducedserum soluble E- and P-selectinchanges in AVM model were shown in Figure 7 Soluble E-and P-selectin were inducible by irradiation Serum levelsof soluble E-selectin doubled at 6 hours after irradiationand returned to baseline at 24 hours Soluble P-selectinwas increased by 5-fold and 4-fold 24 and 48 hours afterirradiation respectively Such high serum levels of solubleP-selectin returned to baseline at 120 hours after radiationRadiosurgery induced serum soluble ICAM-1 and VCAM-1 changes in AVM model were shown in Figure 8 SolubleICAM-1 was increased by 57 plusmn 2 in serum 6 hour afterradiation (119875 lt 05) and doubled within another 6 hoursbefore returning to the baseline at 120 hours Soluble VCAM-1 was increased by 63 plusmn 1 in serum 6 hours after irradiation(119875 lt 05) doubled within another 6 hours andmaintained at48plusmn1 increase at 24 hours before returning to the baseline at120 hours Radiosurgery induced plasma soluble tissue factorchanges in AVM model were shown in Figure 9 Soluble


0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


E-selectin

minus6

(a)

0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


P-selectin

minus6

r = 08550 P lt 002

(b)

Figure 4 Radiosurgery induced E- and P-selectin gene expression in human cerebral microvascular endothelial cells (a) E-selectin geneexpression was doubled at 6 12 24 48 72 and 120 hours after radiosurgery (119875 lt 01) (b)There was a positive correlation between P-selectingene expression and time over 120 hours after radiosurgery (119903 = 0855 119875 lt 02)

tissue factor was increased by 110plusmn6 76plusmn4 and 42plusmn4in plasma at 6 12 and 24 hour after irradiation (119875 lt 05)respectively before returning to the baseline at 120-hour

4 Discussion

Based on our previous immunohistochemistry and transmis-sion electron microscopy studies that radiosurgery inducesendothelial molecular changes in human cerebral AVMspecimens an animal model of AVMs and a mouse cere-bral endothelial cell model [25ndash31] we hypothesize thatendothelial molecules could be upregulated at transcriptionallevel detectable in serum or plasma after radiosurgery andpossibly serve as early predictors for whether AVM vesselsare responsive to radiosurgery treatment The current studyhas achieved the following findings

(i) Human cerebral microvascular endothelial cells aremuch more sensitive to irradiation than thosemicrovessels dilated to form an AVM nidus followingarteriovenous fistula formation in rats

(ii) Expression of E- and P-selectin ICAM-1 PECAM-1 and VCAM-1 is upregulated by radiation at tran-scriptional level and in a dose-dependent manner(Figure 3) Gene expression of E- and P-selectin andICAM-1 are more sensitive to irradiation than thatof PECAM-1 and VCAM-1 Radiosurgery inducedgene expression of P-selectin ICAM-1 PECAM-1andVCAM-1 is linearly correlated to time (Figures 4ndash6)

(iii) Radiosurgery induces significant elevation of serumsoluble E- and P-selectin ICAM-1 VCAM-1 andplasma soluble tissue factor in a rat model of AVMsTiming of the measurement of soluble endothelialmolecules after radiation is critical (Figures 7ndash9)

41 Radiosurgery Induces Inflammatory Molecule ExpressionAlthough a single dose highly focused irradiation reducesthe viability of human cerebral microvascular endothelialcells (Figure 1) and induces apoptosis of AVM vessels ina rat model (Figure 2) vascular endothelial cells remainmetabolically active and able to upregulate their inflamma-tory molecule expression in a dose-dependent (Figure 3)and time-dependent manner (Figures 4ndash6(a)) Radiosurgeryinduced serum concentration changes of E- and P-selectinICAM-1 PECAM-1 and VCAM-1 are time-dependent (Fig-ures 7 and 8) The molecular substrates for radiosurgeryinduced inflammatory response appear to include E- and P-selectin ICAM-1 and VCAM-1 E-selectin acting togetherwith P-selectin mediates the slow rolling of leukocyteson the endothelium leading to firm adhesion [36 37]VCAM-1 plays an important role in lymphocyte homingand migration [17] E-selectin ICAM-1 and VCAM-1 arestimulated by other factors that are present in the AVMvessels [38] These stimuli may act synergistically with radia-tion to further up-regulate E-selectin ICAM-1 and VCAM-1 thereby increasing the specificity of expression Upregu-lation of E- and P-selectin ICAM-1 and VCAM-1 is theearliest demonstrable effects of radiation They collectivelymediate the rolling arrest and transmigration of leukocyteson the endothelium of AVM vessels [25 30] Radiosurgeryinduced inflammatory response plays an important part ina multitude of processes including endothelial cell deathcellular neoproliferation and thrombosis in AVM vessels[39]

42 Radiosurgery Induces Thrombotic Molecule ExpressionRadiosurgery induces thrombus formation in human AVMvessels [40 41] Radiation may influence the expressionof a number of molecules that play important roles in


0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


ICAM-1

r = 07282 P lt 004

minus6

(a)Re

lativ

e exp

ress

ion

()

VCAM-1

0

100

200

300

12 30 48 66 84 102 120minus6

r = 08248 P lt 003


(b)

Figure 5 Radiosurgery induced ICAM-1 and VCAM-1 gene expression in human cerebral microvascular endothelial cells (a) ICAM-1 geneexpression was increased with time over 120 hours after radiosurgery (119903 = 07282 119875 lt 04) (b) There was a positive correlation betweenVCAM-1 gene expression and time over 120 hours after radiosurgery (119903 = 08248 119875 lt 03)

0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


PECAM-1

r = 09520 P lt 001

(a)

0

100

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


vWF

(b)

Figure 6 Radiosurgery induced PECAM-1 and vWF gene expression in human cerebral microvascular endothelial cells (a) PECAM-1 geneexpression was increased with time over 120 hours after radiosurgery (119903 = 0952 119875 lt 01) (b) There was no radiation responsive vWF geneexpression in human cerebral microvascular endothelial cells

coagulation and thrombosis including tissue factor vWFand P-selectin [19 20 42 43] As expected tissue factorgene was not detected in human cerebral microvascularendothelial cells as it does not express on endothelial cells andis normally present on subendothelial cells and in the vascularadventitia of vessels [44] Following radiosurgery of AVMvessels in a rat model vessels lose their endothelial lining[25 26 28] resulting in exposure of subendothelial cells tothe luminal surface which release tissue factor directly to

blood circulation thus plasma levels of soluble tissue factorbecome detectable andwere upregulated in a time-dependentmanner (Figure 9) The presence of exposed tissue factor tothe luminal contents is associated with occlusive thrombusformation [25 26 28] Exposure of subendothelial tissuefactor would be important in the induction of thrombosisin AVM vessels therefore plasma levels of soluble tissuefactor could be an early predictor of the ultimate response toradiosurgery


0

10

20

30

6 12 24 48 72 120minus1

sE-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

(a)

0

100

200

300

400

500

600

700

6 12 24 48 72 120minus1

sP-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

lowast

lowast

lowast

(b)

Figure 7 Radiosurgery induced soluble E- and P-selectin changes in AVM rat serum (a) Serum concentrations of soluble E-selectin weresignificantly increased at 6 and 12 hours after radiosurgery (119875 lt 05) (b) Serum levels of soluble P-selectin were significantly increased at 612 24 48 and 72 hours after radiosurgery (119875 lt 05)

0

25

50

75

6 12 24 48 72 120

sICA

M-1

in se

rum

(ng

mL)

After radiosurgery (hours)minus1

lowast

lowast

(a)

0

5

10

15

20

6 12 24 48 72 120minus1

sVCA

M-1

in se

rum

(ng

mL)


lowast

lowast

(b)

Figure 8 Radiosurgery induced soluble ICAM-1 and VCAM-1 changes in AVM rat serum (a) (b) Serum concentrations of soluble ICAM-1and VCAM-1 were significantly increased at 6 and 12 hours after radiosurgery (119875 lt 05) respectively

The levels of radiosurgery induced endothelial geneexpression and molecular changes observed in the cur-rent study are systematically more accurate than those inirradiated human AVM specimens reported in our previousimmunohistochemistry study [30]The possible explanationsare that human AVM specimens were surgically collectedfrom patients at least 2 years after radiosurgery and theywere not responding to irradiation Soluble forms of vWFand PECAM-1 were not measured as they appear to losethe potential to be an early responsive predictor to radio-surgery Further experiments in AVM patients undergoingradiosurgery may help to delineate the clinical value ofthese endothelial molecules Nevertheless this current studyprovides useful data that may lead to the development of

biomarker for monitoring early responsiveness of radio-surgery

5 Conclusions

This study provides strong evidence that radiosurgeryinduced gene expression of P-selectin ICAM-1 PECAM-1and VCAM-1 in human cerebral microvascular endothelialcells was linearly correlatedwith time and elevation of solubleE- and P-selectin ICAM-1 VCAM-1 and tissue factor inAVMvessels of a ratmodelThus a combination of P-selectinICAM-1 VCAM-1 and tissue factor measured at differenttime points may serve as an early predictor of responsivenessof AVMs to radiosurgery


0

25

50

75

6 12 24 48 72 120

sTiss

ue fa

ctor

in p

lasm

a (pg

mL)

minus1


lowast

lowast

lowast

Figure 9 Radiosurgery induced soluble tissue factor changes inAVM rat plasma Plasma concentrations of soluble tissue factor weredoubled at 6 hours after radiosurgery and maintained at high levelsfor another 18 hours (119875 lt 05) before returning to the baseline

Abbreviations

AVM Arteriovenous malformationsCCA Common carotid arteryCt Threshold cycleEJV External jugular veinELISA Enzyme-linked immunosorbent assayFCS Fetal calf serumFIU Fluorescence intensity unitsGAPDH Glyceraldehyde-3-phosphate dehydrogenaseICAM-1 Intercellular adhesion molecule-1IL-1120573 Interleukin-1 betaMTT 3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

NF120581B Nuclear factor kappa-light-chain-enhancer ofactivated B cells

PECAM-1 Plateletendothelial cell adhesion molecule-1RT-qPCR Real-time quantitative polymerase chain

reactionsVCAM-1 Vascular cell adhesion molecule-1vWF von Willebrand factor

Conflict of Interests

The authors do not have any conflict of interests with thecontent of the paper

Acknowledgment

This work was partially supported by a Macquarie UniversityResearch Development grant to Jian Tu

References

[1] D S Heffez R J Osterdock L Alderete and J Grutsch ldquoTheeffect of incomplete patient follow-up on the reported results ofAVM radiosurgeryrdquo Surgical Neurology vol 49 no 4 pp 373ndash384 1998

[2] B E Pollock J C Flickinger L D Lunsford D J Bis-sonette and D Kondziolka ldquoHemorrhage risk after stereotacticradiosurgery of cerebral arteriovenous malformationsrdquo Neuro-surgery vol 38 no 4 pp 652ndash661 1996

[3] B Karlsson C Lindquist and L Steiner ldquoPrediction of oblit-eration after gamma knife surgery for cerebral arteriovenousmalformationsrdquo Neurosurgery vol 40 no 3 pp 425ndash431 1997

[4] L D Lunsford D Kondziolka J C Flickinger et al ldquoStereotac-tic radiosurgery for arteriovenous malformations of the brainrdquoJournal of Neurosurgery vol 75 no 4 pp 512ndash524 1991

[5] B E Pollock F B Meyer and W A Friedman ldquoRadiosurgeryfor arteriovenous malformationsrdquo Journal of Neurosurgery vol101 no 3 pp 390ndash392 2004

[6] M Yamamoto M Jimbo M Hara I Saito and K MorildquoGamma knife radiosurgery for arteriovenous malformationslong-term follow-up results focusing on complications occur-ring more than 5 years after irradiationrdquo Neurosurgery vol 38no 5 pp 906ndash914 1996

[7] S Lemaire G Lizard S Monier et al ldquoDifferent patterns ofIL-1beta secretion adhesionmolecule expression and apoptosisinduction in human endothelial cells treated with 7alpha-7beta hydroxycholesterol or 7-ketocholesterolrdquo FEBS Lettersvol 440 no 3 pp 434ndash439 1998

[8] TM Carlos and JM Harlan ldquoLeukocyte-endothelial adhesionmoleculesrdquo Blood vol 84 no 7 pp 2068ndash2101 1994

[9] H S Sakhalkar M K Dalal A K Salem et al ldquoLeukocyte-inspired biodegradable particles that selectively and avidlyadhere to inflamed endothelium in vitro and in vivordquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 100 no 26 pp 15895ndash15900 2003

[10] S Quarmby P Kumar and S Kumar ldquoRadiation-inducednormal tissue injury role of adhesion molecules in leukocyte-endothelial cell interactionsrdquo International Journal of Cancervol 82 no 3 pp 385ndash395 1999

[11] R J Shebuski and K S Kilgore ldquoRole of inflammatorymediators in thrombogenesisrdquo Journal of Pharmacology andExperimental Therapeutics vol 300 no 3 pp 729ndash735 2002

[12] A van de Stolpe and P T van der Saag ldquoIntercellular adhesionmolecule-1rdquo Journal of MolecularMedicine vol 74 no 1 pp 13ndash33 1996

[13] D Hallahan J Kuchibhotla and C Wyble ldquoCell adhesionmoleculesmediate radiation-induced leukocyte adhesion to thevascular endotheliumrdquo Cancer Research vol 56 no 22 pp5150ndash5155 1996

[14] D E Jackson ldquoThe unfolding tale of PECAM-1rdquo FEBS Lettersvol 540 no 1ndash3 pp 7ndash14 2003

[15] S M Albelda P D Oliver L H Romer and C A BuckldquoEndoCAM a novel endothelial cell-cell adhesion moleculerdquoJournal of Cell Biology vol 110 no 4 pp 1227ndash1237 1990

[16] G E Rice J M Munro C Corless and M P BevilacqualdquoVascular and nonvascular expression of INCAM-110 A targetfor mononuclear leukocyte adhesion in normal and inflamedhuman tissuesrdquo American Journal of Pathology vol 138 no 2pp 385ndash393 1991

[17] R Alon P D Kassner M W Carr E B Finger M E Hemlerand T A Springer ldquoThe integrin VLA-4 supports tethering and


rolling in flow on VCAM-1rdquo Journal of Cell Biology vol 128 no6 pp 1243ndash1253 1995

[18] B M Chan M J Elices E Murphy and M E HemlerldquoAdhesion to vascular cell adhesion molecule 1 and fibronectinComparison of alpha 4 beta 1 (VLA-4) and alpha 4 beta 7 on thehuman B cell line JYrdquo Journal of Biological Chemistry vol 267no 12 pp 8366ndash8370 1992

[19] N Jahroudi A M Ardekani and J S Greenberger ldquoIonizingirradiation increases transcription of the vonWillebrand factorgene in endothelial cellsrdquo Blood vol 88 no 10 pp 3801ndash38141996

[20] M Verheij L G H Dewit and J A Van Mourik ldquoThe effectof ionizing radiation on endothelial tissue factor activity and itscellular localizationrdquo Thrombosis and Haemostasis vol 73 no5 pp 894ndash895 1995

[21] Z M Ruggeri ldquoStructure and function of von WillebrandfactorrdquoThrombosis and Haemostasis vol 82 no 2 pp 576ndash5841999

[22] J H Jandl Blood Textbook of Hematology Little Brown andCompany Boston Mass USA 1st edition 1996

[23] Z M Ruggeri ldquoVon Willebrand factorrdquo Current Opinion inHematology vol 10 no 2 pp 142ndash149 2003

[24] K G Mann ldquoThrombin formationrdquo Chest vol 124 supplement3 pp 4Sndash10S 2003

[25] K P Storer J Tu M A Stoodley and R I Smee ldquoExpression ofendothelial adhesion molecules after radiosurgery in an animalmodel of arteriovenous malformationrdquo Neurosurgery vol 67no 4 pp 976ndash983 2010

[26] A Karunanyaka J Tu A Watling K P Storer A WindsorandM A Stoodley ldquoEndothelial molecular changes in a rodentmodel of arteriovenous malformation laboratory investiga-tionrdquo Journal of Neurosurgery vol 109 no 6 pp 1165ndash11722008

[27] S Liu V Sammons J Fairhall et al ldquoMolecular responsesof brain endothelial cells to radiationrdquo Journal of ClinicalNeuroscience vol 19 no 8 pp 1154ndash1158 2012

[28] K P Storer A Karunanayaka J Tu R Smee A Watlingand M A Stoodley ldquoIdentification of potential moleculartargets for directed thrombosis after radiosurgery in an animalarteriovenous malformation modelrdquo Neurosurgery vol 58 no2 pp 403ndash404 2006

[29] K P Storer J Tu A Karunanayaka M K Morgan and M AStoodley ldquoThromboticmolecule expression in cerebral vascularmalformationsrdquo Journal of Clinical Neuroscience vol 14 no 10pp 975ndash980 2007

[30] K P Storer J Tu A Karunanayaka M K Morgan and MA Stoodley ldquoInflammatory molecule expression in cerebralarteriovenous malformationsrdquo Journal of Clinical Neurosciencevol 15 no 2 pp 179ndash184 2008

[31] J TuMA StoodleyMKMorgan andK P Storer ldquoResponsesof arteriovenousmalformations to radiosurgery ultrastructuralchangesrdquo Neurosurgery vol 58 no 4 pp 749ndash758 2006

[32] Z Hu Y Xing Y Qian X et al ldquoAnti-radiation damage effectof polyethylenimine as a toll-like receptor 5 targeted agonistrdquoJournal of Radiation Research vol 54 no 2 pp 243ndash250 2013

[33] National Health and Medical Research Council ldquoAustralianCode of Practice for the Care and Use of Animals forScientific Purposesrdquo 7th Edition 2004 httpwwwnhmrcgovau files nhmrcpublicationsattachmentsea16pdf

[34] K P Storer J Tu A Karunanayaka et al ldquoCoadministration oflow-dose lipopolysaccharide and soluble tissue factor induces

thrombosis after radiosurgery in an animal arteriovenous mal-formationmodelrdquoNeurosurgery vol 61 no 3 pp 604ndash611 2007

[35] J L Hintze ldquoAnalysis of variancerdquo in Number Cruncher Sta-tistical Systems (NCSS) 97-Userrsquos Guide-I pp 205ndash278 UTKaysville Utah USA 1997

[36] M P Bevilacqua and R M Nelson ldquoSelectinsrdquo Journal ofClinical Investigation vol 91 no 2 pp 379ndash387 1993

[37] K Ley ldquoThe role of selectins in inflammation and diseaserdquoTrends in Molecular Medicine vol 9 no 6 pp 263ndash268 2003

[38] I Kim S Moon S H Kim H J Kim Y S Koh and G Y KohldquoVascular endothelial growth factor expression of intercellularadhesionmolecule 1 (ICAM-1) vascular cell adhesionmolecule1 (VCAM-1) and E-selectin through nuclear factor-kappa Bactivation in endothelial cellsrdquo Journal of Biological Chemistryvol 276 no 10 pp 7614ndash7620 2001

[39] G T Szeifert M Levivier J Lorenzoni et al ldquoMorpholog-ical observations in brain arteriovenous malformations aftergamma knife radiosurgeryrdquo Progress in Neurological Surgeryvol 27 pp 119ndash129 2013

[40] S D Chang D L Shuster G K Steinberg R P Levyand K Frankel ldquoStereotactic radiosurgery of arteriovenousmalformations pathologic changes in resected tissuerdquo ClinicalNeuropathology vol 16 no 2 pp 111ndash116 1997

[41] B F Schneider D A Eberhard and L E Steiner ldquoHistopathol-ogy of arteriovenous malformations after gamma knife radio-surgeryrdquo Journal of Neurosurgery vol 87 no 3 pp 352ndash357 1997

[42] M Hauer-Jensen L M Fink and J Wang ldquoRadiation injuryand the protein C pathwayrdquo Critical Care Medicine vol 32supplement 5 pp S325ndashS330 2004

[43] M Verheij L G H Dewit M N Boomgaard H-J MBrinkman and J A Van Mourik ldquoIonizing radiation enhancesplatelet adhesion to the extracellular matrix of human endothe-lial cells by an increase in the release of von Willebrand factorrdquoRadiation Research vol 137 no 2 pp 202ndash207 1994

[44] R Lorenzet E Napoleone A Celi G Pellegrini and A DiSanto ldquoCell-cell interaction and tissue factor expressionrdquo BloodCoagulation and Fibrinolysis vol 9 supplement 1 pp S49ndashS591998

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


cells [7] Expression of adhesion molecules including E-selectin P-selectin ICAM-1 PECAM-1 and VCAM-1 isupregulated following irradiation [8] E-selectin is exclusivelyexpressed on the surface of activated endothelial cells [8]and is a critical mediator of leukocyte rollingmdashthe first stepin the cascade of events that lead to firm adhesion andtransmigration into the inflamed tissues [9] The mechanismof induction of E-selectin expression is likely to be via acti-vation of NF120581B by reactive oxygen intermediates generatedby irradiation [10] P-selectin is stored in the Weibel-Paladebodies of endothelial cells Like E-selectin P-selectin is acentral mediator of leukocyte rolling during inflammationand is also able to mediate the adherence of platelets toneutrophils and monocytes [11] allowing this molecule toparticipate in the cross-talk between the inflammatory andthrombogenic processes In addition to its role during inflam-mation P-selectin also participates in coagulation by bindingwith tissue factor to accelerate the formation and depositionof fibrin during thrombogenesis [11] ICAM-1 is constitu-tively expressed on the cell surface of endothelial cells andleukocytes and functionally activates leukocyte-endothelialadhesion and migration [12] The mechanism of inductionof ICAM-1 appears to be similar to that of E-selectin withactivation of NF120581B upregulating transcription of the ICAM-1 gene [13] The expression of PECAM-1 is restricted to cellsof the vascular system The molecule is highly expressed atthe lateral junctions of endothelial cells and on the surfaceof platelets and leukocytes [14] PECAM-1 is required inthe initial formation of endothelial cell-cell contacts [15]and involved in thrombosis [15] VCAM-1 is constitutivelyexpressed on endothelial cells [16] andmediates tethering androlling of lymphocytes [17] and monocytes [18]

Similar to the process of endothelial apoptosis leading toaltered inflammatory properties the coagulative propertiesare also upregulated via increased expression of thromboticmolecules including von Willebrand factor (vWF) [19] andtissue factor [20] vWF is synthesized in endothelial cells andstored in Weibel-Palade bodies Radiation increases consti-tutive synthesis of vWF and exocytosis of the Weibel-Paladebodies Endothelial secretion is the principal source of vWFin the plasma vWF plays a critical role in platelet adhesionand coagulation as well as being important in leukocyterecruitment during inflammation [21] Circulating vWF actsas a carrier protein for factor VIII and prolongs the half-lifeof factor VIII in the circulation by inhibiting the inactivationof factor VIII by factor Xa [22] vWF that is released duringthrombus formation prevents rapid clearance of factor VIIIfrom the plasma thereby promoting thrombin generation[23] Tissue factor is the primary initiator of the extrinsiccoagulation cascade and it is not expressed on endothelialcells but present on subendothelial cells and in the vascularadventitia Under normal conditions tissue factor is anatom-ically sequestered from the blood In response to endothelialdamage or cytokine stimulation tissue factor is activated [24]and forms a complex with factor VIIaThe tissue factor-factorVIIa complex catalyses the activation of factor IX and factorX leading to the conversion of prothrombin to thrombin andthence to the production of fibrin from fibrinogen [22 24]Both blood-borne tissue factor and tissue factor present in the

vessel wall contribute to the initiation and propagation of theprocesses Tissue factor-rich microparticles circulate withinthe blood and serve as a major source of blood-borne tissuefactor [11]

Based on our previous work that endothelial molecularchanges observed in human cerebral AVMs an animalmodelof AVMs and a murine endothelial cell model in responseto radiosurgery [25ndash31] we hypothesize that expression ofendothelial molecules could be upregulated at transcriptionallevel and detectable in blood after radiosurgery The objec-tives of this study were to test our hypothesis in a humancerebral microvascular endothelial cell model as no biopsyhuman AVM specimens that well responded to radiosurgerytreatment are possibly obtained and an AVM animal modelto establish a time course of endothelial molecule changesand identify early predictors of the ultimate response toradiosurgery

2 Materials and Methods

21 Chemicals Primers Reagents andCell Culture All chem-icals M199 and RPMI1640 phenol red free media and cellculture supplements were purchased from Sigma-Aldrich (StLouisMOUSA) unless otherwise specifiedAll primersweresynthesized by Sigma-Aldrich Fetal calf serum (FCS) andSuperscript kit were purchased from Invitrogen (CarlsbadCA USA) Endothelium growth factor and MTT assay kitwere purchased from Roche Applied Science (Indianapo-lis IN USA) RNeasy total RNA extraction mini kit waspurchased from QIAGEN (Hilden Germany) SYBR greenreactionmaster mix was purchased fromApplied Biosystems(Darmstadt Germany) Rat E- and P-selectins ICAM-1PECAM-1 VCAM-1 vonWillebrand factor and tissue factorELISA kits were purchased from Uscn Life Science andTechnology (Wuhan China) Human cerebral microvascularendothelial cells were purchased from Applied BiologicalMaterials (Richmond BC Canada) and maintained in M199medium supplemented with endothelium growth factor and10 FCS in a humidified atmosphere of 37∘C and 5 CO

2

Anti-rat caspase-3 antibody was purchased from Abcam(Cambridge UK) Alexa Fluor 488 conjugated anti-rabbitsecondary antibody was purchased from Molecular Probes(Eugene OR USA)

22 Cell Viability Assay following Irradiation To evaluateradiation effect on cell viability and select irradiation dosefor the following experiments human cerebral microvascularendothelialcells were examined by MTT assay followingirradiation as previously reported [27 32] Briefly humancerebral microvascular endothelial cells were seeded in 96well plates coated with haemaccel (Boehringer PharmaAmsterdam Netherlands) at a density of 8 times 103 for 48 hoursWhen cells were 60 confluent the cellmediumwas replacedby RPMI1640 phenol red free medium supplemented with10 FCS and endothelium growth factor The cell cultureswere then exposed to radiation delivered by an X-Knife linearaccelerator (Radionics Burlington MA USA) with a dose of


















3 Results


0

50

100

Cel

l via

bilit

y (

)


0 Gy5 Gy

15 Gy25 Gy

6 12 24 72 120

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast





0

100

200

300

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43

Inte

nsity

()


Caspase-3

r = 06447 P lt 004





0

100

200

300

Relat

ive e

xpre

ssio

n (

)



PECAM-1VCAM-1vWF

0 5 15 25

lowast

lowast

lowast

lowastlowast


lowast lowast






0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


E-selectin

minus6

(a)

0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


P-selectin

minus6

r = 08550 P lt 002

(b)



4 Discussion








0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


ICAM-1

r = 07282 P lt 004

minus6

(a)Re

lativ

e exp

ress

ion

()

VCAM-1

0

100

200

300

12 30 48 66 84 102 120minus6

r = 08248 P lt 003


(b)


0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


PECAM-1

r = 09520 P lt 001

(a)

0

100

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


vWF

(b)





0

10

20

30

6 12 24 48 72 120minus1

sE-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

(a)

0

100

200

300

400

500

600

700

6 12 24 48 72 120minus1

sP-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

lowast

lowast

lowast

(b)


0

25

50

75

6 12 24 48 72 120

sICA

M-1

in se

rum

(ng

mL)


lowast

lowast

(a)

0

5

10

15

20

6 12 24 48 72 120minus1

sVCA

M-1

in se

rum

(ng

mL)


lowast

lowast

(b)




5 Conclusions



0

25

50

75

6 12 24 48 72 120

sTiss

ue fa

ctor

in p

lasm

a (pg

mL)

minus1


lowast

lowast

lowast


Abbreviations








Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































3 Results


0

50

100

Cel

l via

bilit

y (

)


0 Gy5 Gy

15 Gy25 Gy

6 12 24 72 120

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast





0

100

200

300

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43

Inte

nsity

()


Caspase-3

r = 06447 P lt 004





0

100

200

300

Relat

ive e

xpre

ssio

n (

)



PECAM-1VCAM-1vWF

0 5 15 25

lowast

lowast

lowast

lowastlowast


lowast lowast






0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


E-selectin

minus6

(a)

0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


P-selectin

minus6

r = 08550 P lt 002

(b)



4 Discussion








0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


ICAM-1

r = 07282 P lt 004

minus6

(a)Re

lativ

e exp

ress

ion

()

VCAM-1

0

100

200

300

12 30 48 66 84 102 120minus6

r = 08248 P lt 003


(b)


0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


PECAM-1

r = 09520 P lt 001

(a)

0

100

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


vWF

(b)





0

10

20

30

6 12 24 48 72 120minus1

sE-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

(a)

0

100

200

300

400

500

600

700

6 12 24 48 72 120minus1

sP-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

lowast

lowast

lowast

(b)


0

25

50

75

6 12 24 48 72 120

sICA

M-1

in se

rum

(ng

mL)


lowast

lowast

(a)

0

5

10

15

20

6 12 24 48 72 120minus1

sVCA

M-1

in se

rum

(ng

mL)


lowast

lowast

(b)




5 Conclusions



0

25

50

75

6 12 24 48 72 120

sTiss

ue fa

ctor

in p

lasm

a (pg

mL)

minus1


lowast

lowast

lowast


Abbreviations








Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43

Inte

nsity

()


Caspase-3

r = 06447 P lt 004





0

100

200

300

Relat

ive e

xpre

ssio

n (

)



PECAM-1VCAM-1vWF

0 5 15 25

lowast

lowast

lowast

lowastlowast


lowast lowast






0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


E-selectin

minus6

(a)

0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


P-selectin

minus6

r = 08550 P lt 002

(b)



4 Discussion








0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


ICAM-1

r = 07282 P lt 004

minus6

(a)Re

lativ

e exp

ress

ion

()

VCAM-1

0

100

200

300

12 30 48 66 84 102 120minus6

r = 08248 P lt 003


(b)


0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


PECAM-1

r = 09520 P lt 001

(a)

0

100

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


vWF

(b)





0

10

20

30

6 12 24 48 72 120minus1

sE-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

(a)

0

100

200

300

400

500

600

700

6 12 24 48 72 120minus1

sP-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

lowast

lowast

lowast

(b)


0

25

50

75

6 12 24 48 72 120

sICA

M-1

in se

rum

(ng

mL)


lowast

lowast

(a)

0

5

10

15

20

6 12 24 48 72 120minus1

sVCA

M-1

in se

rum

(ng

mL)


lowast

lowast

(b)




5 Conclusions



0

25

50

75

6 12 24 48 72 120

sTiss

ue fa

ctor

in p

lasm

a (pg

mL)

minus1


lowast

lowast

lowast


Abbreviations








Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


E-selectin

minus6

(a)

0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


P-selectin

minus6

r = 08550 P lt 002

(b)



4 Discussion








0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


ICAM-1

r = 07282 P lt 004

minus6

(a)Re

lativ

e exp

ress

ion

()

VCAM-1

0

100

200

300

12 30 48 66 84 102 120minus6

r = 08248 P lt 003


(b)


0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


PECAM-1

r = 09520 P lt 001

(a)

0

100

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


vWF

(b)





0

10

20

30

6 12 24 48 72 120minus1

sE-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

(a)

0

100

200

300

400

500

600

700

6 12 24 48 72 120minus1

sP-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

lowast

lowast

lowast

(b)


0

25

50

75

6 12 24 48 72 120

sICA

M-1

in se

rum

(ng

mL)


lowast

lowast

(a)

0

5

10

15

20

6 12 24 48 72 120minus1

sVCA

M-1

in se

rum

(ng

mL)


lowast

lowast

(b)




5 Conclusions



0

25

50

75

6 12 24 48 72 120

sTiss

ue fa

ctor

in p

lasm

a (pg

mL)

minus1


lowast

lowast

lowast


Abbreviations








Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)


ICAM-1

r = 07282 P lt 004

minus6

(a)Re

lativ

e exp

ress

ion

()

VCAM-1

0

100

200

300

12 30 48 66 84 102 120minus6

r = 08248 P lt 003


(b)


0

100

200

300

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


PECAM-1

r = 09520 P lt 001

(a)

0

100

12 30 48 66 84 102 120

Relat

ive e

xpre

ssio

n (

)

minus6


vWF

(b)





0

10

20

30

6 12 24 48 72 120minus1

sE-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

(a)

0

100

200

300

400

500

600

700

6 12 24 48 72 120minus1

sP-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

lowast

lowast

lowast

(b)


0

25

50

75

6 12 24 48 72 120

sICA

M-1

in se

rum

(ng

mL)


lowast

lowast

(a)

0

5

10

15

20

6 12 24 48 72 120minus1

sVCA

M-1

in se

rum

(ng

mL)


lowast

lowast

(b)




5 Conclusions



0

25

50

75

6 12 24 48 72 120

sTiss

ue fa

ctor

in p

lasm

a (pg

mL)

minus1


lowast

lowast

lowast


Abbreviations








Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

6 12 24 48 72 120minus1

sE-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

(a)

0

100

200

300

400

500

600

700

6 12 24 48 72 120minus1

sP-s

elec

tin in

seru

m (n

gm

L)


lowast

lowast

lowast

lowast

lowast

(b)


0

25

50

75

6 12 24 48 72 120

sICA

M-1

in se

rum

(ng

mL)


lowast

lowast

(a)

0

5

10

15

20

6 12 24 48 72 120minus1

sVCA

M-1

in se

rum

(ng

mL)


lowast

lowast

(b)




5 Conclusions



0

25

50

75

6 12 24 48 72 120

sTiss

ue fa

ctor

in p

lasm

a (pg

mL)

minus1


lowast

lowast

lowast


Abbreviations








Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

25

50

75

6 12 24 48 72 120

sTiss

ue fa

ctor

in p

lasm

a (pg

mL)

minus1


lowast

lowast

lowast


Abbreviations








Acknowledgment


References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















in vitro and in vivo models of

Documents