DTIC ADA544582: Timing of Captopril Administration Determines Radiation Protection or Radiation Sensitization in a Murine Model of Total Body Irradiation

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by  Defense Technical Information Center

ExperimentalHematology2010;38:270–281
Timing of captopril administration determines radiation protection
or radiation sensitization in a murine model of total body irradiation
Thomas A. Davisa, Michael R. Landauerb, Steven R. Mogb,
Michal Barshishat-Kupperc, Stephen R. Zinsa, Mihret F. Amarea, and Regina M. Dayc
aDepartmentofRegenerativeMedicine,NavalMedicalResearchCenter,SilverSpring,
Md.,USA;bArmedForcesRadiobiologyResearchInstitute,UniformedServicesUniversityoftheHealthSciences,
Bethesda,Md.,USA;cDepartmentofPharmacology,UniformedServicesUniversityoftheHealthSciences,Bethesda,Md.,USA
(Received5January2010;revised5January2010;accepted20January2010)
Objective. Angiotensin II (Ang II), a potent vasoconstrictor, affects the growth and develop-
ment of hematopoietic cells. Mixed findings have been reported for the effects of
angiotensin-converting enzyme (ACE) inhibitors on radiation-induced injury to the hemato-
poietic system. We investigated the consequences of different regimens of the ACE inhibitor
captopril onradiation-induced hematopoietic injury.
Materials and Methods. C57BL/6 mice were either sham-irradiated or exposed to 60Co total
bodyirradiation(0.6Gy/min).Captoprilwasprovidedinthewaterfordifferenttimeperiods
relativetoirradiation.
Results. Inuntreatedmice,thesurvivalratefrom7.5Gywas50%at30dayspostirradiation.
Captopril treatment for7 dayspriortoirradiationresulted inradiosensitizationwith100%
lethalityandarapiddeclineinmaturebloodcells.Incontrast,captopriltreatmentbeginning
1hourpostirradiationandcontinuingfor30daysresultedin100%survival,withimproved
recoveryofmaturebloodcellsandmultilineagehematopoieticprogenitors.Innonirradiated
controlmice,captoprilbiphasicallymodulatedLinLmarrowprogenitorcellcycling.After2
days,captoprilsuppressedG LG transitionandagreaternumberofcellsenteredaquies-
0 1
centstate.However,after7daysofcaptopriltreatmentLinLprogenitorcellcyclingincreased
comparedto untreated controlmice.
Conclusion. These findings suggest that ACE inhibition affects hematopoietic recovery
following radiation by modulating the hematopoietic progenitor cell cycle. The timing of
captopril treatment relative to radiation exposure differentially affects the viability and
repopulation capacityof spared hematopoietic stem cells and, therefore, can result in either
radiation protection or radiation sensitization. Published by Elsevier Inc. on behalf of the
ISEH - Society for HematologyandStem Cells.
High-dose total body irradiation (TBI) as the result of and microvascular hemorrhage in vital organs, such as
a nuclear accident, terrorist event, or as a clinical therapy the brain and gastrointestinal (GI) tract. Of clinical impor-
for cancer, has significant hematopoietic toxicity. TBI tance, severe and prolonged cytopenia is a major cause
destroys the hematopoietic stem cells (HSC) in the bone of morbidity and mortality following myeloablation and
marrow compartment, which are critical for blood cell HSC transplantation in the clinical setting [2].
regeneration[1].FollowinglethalTBItothehematopoietic The sensitivity of the hematopoietic system to radiation
system,mortalityinmicetypicallyoccursbetween2and4 isbelievedtobeduetotherelativelyrapidcyclingtimesof
weeks postirradiation.Deathresultsfromimmunefunction bothshort-termandlong-termreconstitutinghematopoietic
impairment, infection, and increased vascular permeability stem cells (ST-HSC and LT-HSC, respectively) [3,4].
Compared with quiescent or slowly cycling cells, rapidly
cycling cells are predisposed to increased DNA damage
Offprint requests to: Regina M. Day, Ph.D., Department of Pharma-
from radiation exposure, resulting in higher levels of
cology,UniformedServicesUniversityoftheHealthSciences,BuildingC,
apoptosisorsenescence[5].Agentsthatinducequiescence
Room2023,4301JonesBridgeRoad,Bethesda,MD20814-4799;E-mail:
[email protected] of the HSC population inhibit HSC apoptosis and
0301-472X/10$–seefrontmatter.PublishedbyElsevierInc.onbehalfoftheISEH-SocietyforHematologyandStemCells.
doi: 10.1016/j.exphem.2010.01.004
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4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Timing Of Captopril Administration Determines Radiation Protection Or
5b. GRANT NUMBER
Radiation Sensitization In A Murine Model Of Total Body Irradiation
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6. AUTHOR(S) 5d. PROJECT NUMBER
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Naval Medical Research Center,Department of Regenerative REPORT NUMBER
Medicine,Silver Spring,MD,20910
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13. SUPPLEMENTARY NOTES
Experimental Hematology, Volume 38, Issue 4, Pages 270-281 (April 2010)
14. ABSTRACT
Objective. Angiotensin II (Ang II), a potent vasoconstrictor, affects the growth and development of
hematopoietic cells. Mixed findings have been reported for the effects of angiotensin-converting enzyme
(ACE) inhibitors on radiation-induced injury to the hematopoietic system. We investigated the
consequences of different regimens of the ACE inhibitor captopril on radiation-induced hematopoietic
injury. Materials and Methods. C57BL/6 mice were either sham-irradiated or exposed to 60Co total body
irradiation (0.6 Gy/min). Captopril was provided in the water for different time periods relative to
irradiation. Results. In untreated mice, the survival rate from 7.5 Gy was 50% at 30 days postirradiation.
Captopril treatment for 7 days prior to irradiation resulted in radiosensitization with 100% lethality and a
rapid decline in mature blood cells. In contrast, captopril treatment beginning 1 hour postirradiation and
continuing for 30 days resulted in 100% survival, with improved recovery of mature blood cells and
multilineage hematopoietic progenitors. In nonirradiated control mice, captopril biphasically modulated
LinL marrow progenitor cell cycling. After 2 days, captopril suppressed G0LG1 transition and a greater
number of cells entered a quiescent state. However, fter 7 days of captopril treatment LinL progenitor cell
cycling increased compared to untreated control mice. Conclusion. These findings suggest that ACE
inhibition affects hematopoietic recovery following radiation by modulating the hematopoietic progenitor
cell cycle. The timing of captopril treatment relative to radiation exposure differentially affects the viability
and repopulation capacity of spared hematopoietic stem cells and, therefore, can result in either radiation
protection or radiation sensitization. Published by Elsevier Inc. on behalf of the ISEH - Society for
Hematology and Stem Cells.
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T.A.Davisetal./ExperimentalHematology2010;38:270–281 271
senescenceandtherebypreventradiation-inducedstemcell radiation exposure. Mice administered captopril for 7
pool exhaustion. Our laboratory has shown that the isofla- consecutivedays prior toirradiation exhibitedradiosensiti-
vone genistein transiently arrests the LT-HSC in the G / zation,whiletreatmentsthatbeganasearlyas1houror24
0
G phases of the cell cycle and reduces radiation-induced hours after irradiation were protective. The sensitizing vs
1
genotoxicity, senescence, and stem cell pool exhaustion protective effects of the two types of regimens were
[6,7]. However, prevention of mortality from radiation- reflected in the severity of radiation-induced weight loss
induced hematopoietic injury can also be achieved by and in the repopulation rates of hematopoietic progenitor
agents that promote proliferation of the ST-HSC to cells.
replenish mature blood cells, although at the transient
expenseofreducedLT-HSCpools.Theradiationprotective
agents granulocyte-colony stimulating factor, interleukins,
Materials and methods
and thrombopoietin, for example, increase proliferation
and differentiation of ST-HSC and promote mature blood Experimental design
cell repopulation [4,8,9]. Female C57BL/6J mice (The Jackson Laboratory, Bar Harbor,
Therenin-angiotensinsystemiscriticalforregulationof ME, USA) were 12 to 14 weeks of age (17.5(cid:2)21.5 g) at the
time of irradiation. Mice were housed in groups of four to five
blood pressure and blood volume homeostasis [10], but
percageinafacilityaccreditedbytheAssociationforAssessment
components of this system also regulate proliferation and
and Accreditation of Laboratory Animal Care International.
maturation of hematopoietic cells. Angiotensin II (Ang II)
Animal rooms were maintained at 21(cid:3)C 6 2(cid:3)C, 50% 6 10%
directly modulates developmentandproliferation ofhema-
humidity,and12-hourlight/darkcycle.Commercialrodentration
topoietic progenitor cells (HPC) through Ang II receptors (HarlanTekladRodentDiet8604,HarlanLaboratories,Madison,
on the surfaces of these cells [11–14]. Plasma levels of WI, USA) and acidified water (pH 2.5(cid:2)3.0), to control opportu-
Ang II are tightly regulated, and the protease angiotensin- nistic infections [21], were freely available. All animal handling
converting enzyme (ACE) is required for the maturation procedures were performed in compliance with guidelines from
of Ang II from its inactive precursor angiotensin I. Inhibi- theNationalResearchCouncilandwereapprovedbytheInstitu-
tion of ACE blocks the formation of activeAng II and can tional Animal Care and Use Committee of the Armed Forces
RadiobiologyResearch Institute(Bethesda,MD, USA).
reversibly inhibit HSC proliferation in cell culture and in
Na¨ıve mice were randomized and assigned to groups that
vivo [14–16].
receivedeithernotreatmentorvariousregimensofcaptopriltreat-
The literature provides mixed reports for the effects of
ment. We previously determined that 7.5-Gy TBI results in 50%
AngIIandACEinhibitorsonradiation-inducedhematopoi-
lethality within 30 days (LD ) for C57BL/6J mice in Armed
50/30
eticinjury.MiceadministeredAngIIfor2to7daysbegin- Forces Radiobiology Research Institute’s 60Co radiation facility
ning the day of irradiation exhibited increased 30-day [6].Asdescribedpreviously[22],miceinthecurrentexperiments
survival and improved white blood cell recovery [17,18], received TBI at 0.6 Gy/min. Control mice were sham-irradiated.
presumably through increased proliferation and self- Captopril (USP grade; Sigma-Aldrich, St Louis, MO, USA) was
renewal of spared multilineage hematopoietic stem and dissolved in acidified water at 0.55 g/L. A previous study estab-
progenitor cells. Paradoxically, positive results have also lishedthestabilityofcaptoprilinacidifiedwater[23].Theeffect
been reported for hematopoietic radiation protection by ofcaptoprilonwaterintakewasmonitoredfor30days,withand
withoutirradiation(Fig.1).Captoprilresultedinanincreaseinthe
ACE inhibitors. Early studies showed that the ACE inhib-
daily volume of water consumed by nonirradiated mice (control
itor captopril failed to provide bone marrow protection in
3.20 6 0.03 mL/day vs captopril treatment 4.00 6 0.20 mL/
rats when administration was initiated 7 days prior to irra-
day). Radiation exposure resulted in a reduction in the average
diation and continued for 28 days after irradiation [19].
waterconsumptionforapproximately2weeksandwasmitigated
However, perindopril, another ACE inhibitor, increased
by captopril (Fig. 1). The average captopril consumption was
30-day survival and protected ST-HSC when mice were calculatedbasedonvolumesofwaterconsumedandbodyweights
treated for only 4 consecutive days beginning 2 days prior duringthetimecourseoftheexperiments.Nonirradiatedmicehad
to irradiation through 2 days postirradiation [20]. This aneffectivedosageof0.1160.01mg/kg/day.Inirradiatedmice
protection was shown to be due to the inhibition of Ang given captopril prior toirradiation, theaverage consumption was
II maturation, because inhibitors of the Ang II type I 0.1060.02mg/kg/day.Inmicetreatedwithcaptoprilafterirradi-
receptorhadsimilarprotectiveeffectsonthehematopoietic ation,theaverageconsumptionwas0.1060.05mg/kg/day.
For survival studies, mice were randomly assigned to one of
system.
seven groups. Two groups were sham-irradiated (nonirradiated:
In this article, radiosensitization and radioprotection,
handled/manipulatedthesameasirradiatedmice):1)notreatment
respectively, are defined as increased sensitivity or
(n 5 20) and 2) captopril for 30 days (n 5 20). Five groups
increased protection of cells, tissues, or organisms to
received a single dose of 7.5-Gy TBI and were treated with or
gammaradiation,asaresultofanagentbeingadministered
without captopril. The following groups received radiation (day
beforeand/orafterradiationexposure.Wedemonstratethat 0 5 day of irradiation): 3) no treatment (n 5 16), 4) captopril
captoprilcanhaveeitherradiosensitizingorradioprotective for 7 days before irradiation through 30 days postirradiation
effects, depending on the time of administration relativeto (days(cid:2)7throughþ30)(n520),5)captoprilfordays(cid:2)7through
272 T.A.Davisetal./ExperimentalHematology2010;38:270–281
Figure1. Effectofcaptoprilondrinkingwaterconsumption.Volumeofwaterconsumedwasmeasuredeachdayforthefull-timecourseoftheexperiment.
Theestimatedvolumeofwaterconsumedpermousewascalculated.Captoprilwasadministeredpreirradiationfromday(cid:2)7today0(dayofirradiation).
Captopriladministeredpostirradiationwasfromday0(beginning1hourafterirradiation)todayþ30.
0(n520),6)captoprilfordaysþ1(beginning24hoursafterirra- calculated based on the total number of viable, nucleated cells
diation)throughþ7(n520),and7)captoprilfordays0(begin- per femur and on the number of colonies scored per number of
ning1hourafterirradiation)throughþ30(n520).Survivalwas cellsplated.
monitoredfor30daysafterTBI.
For hematological and tissue analysis, micewere randomized Cell-cycle analysis ofLin(cid:2) andLSKþ cells
intooneoffivegroups.Twogroupsweresham-irradiated(nonir- Lin(cid:2) bone marrow cells were isolated using mouse Lin(cid:2) cell
radiated): 1) no treatment and 2) captopril-treated. The dose of magneticcellsorting(MACS)CellSelectionKitsaccordingtomanu-
irradiation for hematology or tissue analysis was either 7.5 Gy facturer’s instructions (Miltenyi Biotec, Auburn, CA, USA). Cell-
or 6 Gy, and groups were: 3) no treatment, 4) captopril for days cycleanalysiswasperformedasdescribedpreviously[22].Lin(cid:2)cells
(cid:2)7through0,and5)captoprilforday0throughthedayoftissue wereincubatedfor30minutesat4(cid:3)CwithR-phycoerythrin(cid:2)Sca-1
harvest. and allophycocyanin-conjugated anti-mouse CD117 (cKit; anti-
Micewere anesthetized with pentobarbital andblood was ob- bodies from BD-Pharmingen, San Jose, CA, USA). After surface
tainedbycardiacpuncture,asdescribedpreviously[6].Complete stainingwascomplete,Lin(cid:2)cellswerewashed,fixedwith0.5mL
bloodcountswithdifferentialswereobtainedusingaBakerAdvia 1.4%paraformaldehydeinphosphate-bufferedsalinefor1hourat
2120 Hematology Analyzer (Siemens, Tarrytown, NY, USA). 4(cid:3)C, and then incubated with an equal volume of 0.2% Triton-X
Separatemicewereusedateachtimepoint(n55(cid:2)6pergroup). overnight. Fixed, permeabilized Lin(cid:2) cells were stained with
For determination of microhemorrhage, gross necropsies were Ki-67(cid:2)fluorescein isothiocyanate (BD Pharmingen) for 2 hours.
performedonmiceatthetimeofdeathoratthestudytermination Cellswerewashedandresuspendedinstainingbuffercontaining2
endpoint(day10orday14)(n56(cid:2)10pergroup).Organs,espe- mg/mL7-aminoactinomycin-D(Sigma).Lin(cid:2)andgatedLSKþcells
ciallytheGItractandbrain,werescreenedforhemorrhage,either (Lin(cid:2) Sca-1þcKitþ) were analyzed for Ki-67 expression and 7-
petechiae or ecchymoses. Tissues with hemorrhage were immer- aminoactinomycin-Dincorporationbyperformingfour-colorparam-
sion fixed in 10% neutral-buffered formalin with ionized zinc etersortingusingaCoulterEliteflowcytometer(Coulter,Hialeah,
(Z-Fix;AnatechLtd,BattleCreek, MI,USA). FL,USA).
Hematopoietic progenitor colony-forming cellassays Statistical analysis
Femoralbonemarrowcellswereisolatedasdescribedpreviously Fisher’s exact test wasused for analysis of survivaldata. Weight
[22].Unfractionatedbonemarrowcellswereplatedinmultipoten- datawereanalyzedusingone-wayanalysisofvarianceandDunn’s
tialmethylcelluloseculturemedium(MethocultGFM3434;Stem- test, using SigmaStat, 3.1 (Point Richmond, CA, USA). Hema-
CellTechnologies,Vancouver,BC,Canada)at1to5(cid:4)104cells tology, clonogenic CFU assays, and cell-cycle results are ex-
perdish.Coloniesderivedfromcolony-formingunitgranulocyte- pressed as the mean 6 standard error of mean. Statistical
erythroid-macrophage-megakaryocyte (CFU-GEMM), colony- significance between the paired results was determined using
forming unit granulocytic-macrophage (CFU-GM), blast-forming one-way analysis of variance followed by Holm-Sidak or Tukey
unit erythroid (BFU-E), and colony-forming unit macrophage postanalysis. A value of p # 0.05 was considered significant.
(CFU-M)werescoredafter8to12daysofincubationinahumid- Mean survival time (MST) of decedents over 30 days was deter-
ifiedenvironment,5%CO asdescribedpreviously[22].Absolute minedbytheKaplan-Meiersurvivalanalysis,usingSingleGroup
2
numbers of clonogenic CFU progenitor cells per femur were statisticsintheSigmaStat,3.1software.
T.A.Davisetal./ExperimentalHematology2010;38:270–281 273
Figure2. Treatmentofmicewithcaptoprilfollowinghigh-doseirradiationisradioprotective,whereastreatmentpriortoirradiationhasaradiosensitization
effect.Micewereuntreatedorwereadministeredcaptopril(0.1mg/kg/day)intheirdrinkingwaterbeforeand/orafterexposureto7.5Gy60Cogammatotal
bodyirradiation(TBI).(A)Thepercentageofmicesurvivingat30daysisshown.Resultsrepresentatotalof16to20micepergroup.*p!0.05from
untreatedirradiatedmice.**p!0.001fromuntreatedirradiatedmice.(B)Graphof30daysurvival.
Results Administration of captopril prior to irradiation resulted in
radiosensitization. When captopril administration began 7
Effectofcaptoprilregimenson30-daysurvivalfollowing days before irradiation (day (cid:2)7) and continued until the
7.5-Gy TBI dayofirradiation(day 0),none (0%) ofthe mice survived,
We previously showed that for female C57BL/6J mice the and the MST was 17 days (95% CI, 16(cid:2)18). Mice pre-
LD was 7.52 Gy (95% confidence interval [CI], treated with captopril for 7 days before irradiation and
50/30
7.44(cid:2)7.59) in our 60Co facility [24]. We used this dose continuing through day 30 postirradiation (day þ30) also
ofirradiationtodeterminetheeffectsofcaptopriltreatment exhibited radiosensitization, with 15% survival (p ! 0.05
either before, after, or before and after irradiation. In compared to untreated irradiated mice) and an MSTof 21
untreated irradiated mice that received 7.5 Gy, 30-day days (95% CI, 20(cid:2)22). In marked contrast, mice that
survival was again 50%. The MST of decedents was 20 received captopril only after irradiation exhibited a protec-
days (95% CI, 15(cid:2)25) (Fig. 2A, B). The timing of tiveeffectagainsthematopoieticinjury.Thirty-daysurvival
mortality was consistent with acute hematopoietic injury. was 85% for mice that were administered captopril
274 T.A.Davisetal./ExperimentalHematology2010;38:270–281
Figure2. (Continued.)(C)Time-dependentchangeinbodyweightinnonirradiatedcontrolmiceormicethatreceivedcaptoprilintheirdrinkingwater
beforeand/orafterexposureto7.5Gy60CoTBI.Datarepresentthechangeinbodyweight6standarderror.(n520micepergroup).Statisticalfindings
arenotedinthetext.
beginning 24 hours after irradiation (day þ1) and We examined the effects of two captopril regimens on
continuing for 7 days (p ! 0.05 from untreated irradiated hematopoieticrecoveryfrom7.5-GyTBI.Groupsofmice
mice). The MST of the three mice that died was 23 days received1)notreatment,2)captoprilfor7dayspriortoirra-
(95% CI, 19(cid:2)26). Mice administered captopril beginning diation(day(cid:2)7through0),3)orcaptoprilafterirradiation
1 hour (day 0) after irradiation and continuing through beginning1hourpostirradiation(day0)andcontinuingfor
day þ30 exhibited 100% survival for 30 days (p ! 0.001 either 2 hours or 2, 7, 10, or 14 days after irradiation.
compared to untreated irradiated mice). Completebloodcountanalyseswereperformedonseparate
The radiation sensitization or protection induced by groupsofmiceat2hours,and2,7,10,and14dayspostir-
captopril was reflected in the body weights of the mice radiation.Arapiddeclineofallmaturebloodcelltypeswas
(Fig. 2C). Radiation in untreated mice resulted in signifi- observed following irradiation (Fig. 3A). In mice that
cant weight loss observable within 1 day postirradiation, received captopril prior to irradiation, a loss of red blood
with a nadir w13 to 20 days postirradiation. Mice that cells occurred earlier and was more pronounced during
received captopril prior to irradiation, either days (cid:2)7 the entire time coursewhen compared to untreated irradi-
through 0 or days (cid:2)7 through þ30, exhibited greater ated mice. The decline in red blood cells was reflected by
weight loss than untreated irradiated mice. In mice admin- decreased levels in hematocrit and hemoglobin compared
istered captopril before irradiation, the reduction in body withuntreatedirradiatedmice(datanotshown).Reticulo-
weight occurred for an extended time, and was significant cyte recovery was also reduced in mice given captopril
between days 16 and 28 postirradiation (p ! 0.05 beforeirradiationandwassignificantonday14postirradi-
compared to untreated irradiated mice). In contrast, mice ation. This modest decline in circulating erythroid cells is
treated with captopril after radiation exposure (days consistentwithsubstantialradiosensitizationofcommitted
0 through þ30) maintained higher average body weights erythroidprogenitorcells.Failureofplateletrecoverywas
compared with untreated irradiated mice. The increased also observed on day 14 postirradiation in the group of
weight was significant between days 9 and 20 when mice receiving captopril from day 7 through day 0. In
compared to untreated irradiated mice (p ! 0.05). Capto- contrast, treatment of mice with captopril starting 1 hour
pril treatment in the absence of radiation had no effect on afterirradiationresultedinreducedradiation-inducedloss
body weight when compared to untreated control mice. of red blood cells, significant on day 14. Administration
of captopril postirradiation also significantly improved
Captopril administration affects mature blood cell reticulocyte recovery on day 14 and platelet recovery on
recovery following irradiation days10to14postirradiation.
We hypothesized that protection from radiation-induced Radiation-induced hematopoietic injury can lead to
hematopoieticinjurybycaptopriltreatmentpostirradiation hemorrhage (petechiae and ecchymoses) and microhemor-
was associated with enhanced hematopoietic progenitor rhage in multiple organs, including the brain and GI tract
cell activity resulting in accelerated blood cell recovery. [4]. Hemorrhages associated with acute radiation injury
T.A.Davisetal./ExperimentalHematology2010;38:270–281 275
Figure 3. Effect of captopril administration on radioprotection. Mice
received no treatment or were administered captopril in their drinking
waterbefore(day(cid:2)7today05dayofirradiation)orafter(day0[starting
1hourafterirradiation]today30)exposureto7.5Gy60Cogammatotal
bodyirradiation.(A)Peripheralbloodwhitebloodcells(WBC),redblood
cells (RBC), reticulocytes, lymphocytes, platelets, and absolute neutro-
phils from irradiatedmice,sampleswere taken at 2 hours(indicated on
day 0), 2, 7, 10, or 14 days postirradiation. Control blood cell levelsin
untreated, nonirradiated mice are also indicated for blood cell types,
except for lymphocytes (3.84 (cid:4) 103 6 0.4) and WBC (4.4 (cid:4) 103 6
0.5). Data show meanvalues 6standard error of mean, n 55(cid:2)6 mice
per group. *p ! 0.05 compared with radiation alone cell counts at the
sametimepoint.(B)Histologicalcomparisonofbrainmicrohemorrhages
in the subcortical cerebrum (100(cid:4) magnification) and cerebellar cortex
(200(cid:4) magnification) of mice at day 14 postirradiation. Representative
photomicrographs of hematoxylin and eosin(cid:2)stained tissue for mice
treatedwithcaptoprilpriorto(upperpanelswithhemorrhage)orfollowing
(lower panels, normal brain) radiation exposure. The microhemorrhages
areindicatedbyarrowsandoccurperiventricularlyinthesubcorticalcere-
brum;ventricles(V)andcorpuscallosum(CC)areidentifiedfororienta-
tion. Microhemorrhages also occurred perivascularly in the cerebellar
cortex extending from thewhite matter (WM) through the granular (G)
andpiriform/Purkinje(P)layers,abuttingthemolecular(M)layer.
areascribedtolossofplateletsbelowathresholdlevel[25]. TBI, there is an immediate proliferative demand on func-
We examined the effects of 7.5 Gy 60Co on brain and GI tionally spared LT- and ST-HSC to repopulate the ablated
hemorrhaging on days 10 and 14 postirradiation. No pete- hematopoietic system [7]. Sustained hyperproliferation
chiae were detected on day 10. However, on day 14, 60% and differentiation signals can result in fewer HSC self-
of untreated irradiated mice had grossly observable brain renewing divisions and can lead to exhaustion of the HSC
hemorrhages (4 were classified as mild; 2 were classified pool and marrow repopulating failure. The molecular
as minimal; n 5 10). The sensitization or protection of mechanisms preserving HSC require a balance between
blood cells by captopril was reflected by the presence or proliferation, differentiation, and self-renewal. These inter-
absence of gross hemorrhage and intracerebral microhe- related processes and mechanisms are important and not
morrhages. All mice treated with captopril prior to irradia- fullyunderstood.Basedonourpostirradiationsurvivalfind-
tion (days (cid:2)7 through 0) exhibited brain hemorrhages (4 ings, we hypothesized that spared HSC are substantially
mild, 6 minimal; n 5 10) (Fig. 3B). In contrast, none of less abundant and/or have an impaired repopulating poten-
the mice treated with captopril postirradiation (starting on tial in mice receiving captopril before irradiation as
day 0) exhibited brain hemorrhages (n 5 6). Evidence of compared to mice treated with captopril postirradiation.
gross vascular or microvascular hemorrhage in the GI tract To assess the kinetics of marrow repopulation, we exam-
was not observed in any group (data not shown). inedtotalbonemarrowcellularityandquantifiedassayable
HPC production (in vitro CFU clonogenic assays) using
Captopril effects on bone marrow recovery marrow obtained from mice treated with radiation alone
Radiation-induced stress can compromise the hematopoi- and in the two captopril-radiation regimens described
etic repopulation potential of HSC. Following high-dose here. Mice that received captopril before irradiation died
276 T.A.Davisetal./ExperimentalHematology2010;38:270–281
withgreaterbonemarrowprogenitor/stemdepletion,which 2daysofcaptopriltreatment.However,asimilarG toG /
1 2
resulted in impaired marrow repopulation and decreased M/S cell-cycle progression was detected in the LSKþ after
survival (radiosensitization). On day 14 postirradiation, 2 or 7 days of captopril treatment (Fig. 5B).
mice receiving captopril prior to irradiation (days (cid:2)7
through 0) exhibited reduced nucleated cellularity in the
femoralbonemarrowcomparedwithmiceintheuntreated Discussion
radiation-only group. This indicated a gross deficit in total Components of the renin-angiotensin system have been
hematopoietic cell production in the marrow compartment investigatedforhematopoieticradiationprotectionbecause
(Fig.4A). Nosignificant differences inthe number oftotal of the role of this system in hematopoietic cell regulation.
nucleated cells were observed between the radiation-only BecauseACEinhibitorsareusedextensivelyinthegeneral
group and the captopril postirradiation treatment groups. population, it is critical to understand the effects of these
Next, we characterized the types of hematopoietic drugs on radiation-induced hematopoietic injury because
progenitors present in each treatment group to assess the radiotherapyisacommontherapeuticmodalityfortreating
rapid loss of CFU and their subsequent expansion in the cancer, leukemia, and lymphoma [28,29]. Paradoxically,
marrow compartment following radiation-induced hemato- both ACE inhibitors and Ang II peptides have been shown
poieticinjury.Atday5,butnotday14,postirradiation,the to provide protection of the hematopoietic system
number of total marrow CFU was significantly reduced in [16,18,20]. Here, we demonstrate that treatment of mice
mice pretreated with captopril when compared with withtheACEinhibitorcaptoprilresultedineitherradiation
untreated irradiated mice (Fig. 4B, C). We also observed protectionorradiationsensitization,dependingonthetime
specific reductions in CFU-M populations. In contrast, of captopril administration. Captopril treatment in nonirra-
mice treated with captopril postirradiation exhibited diatedmicehadabiphasiceffectonthecyclingofST-HSC
a significant increase in myeloid CFU progenitor cell with transient quiescence after 2 days of treatment, fol-
production (CFU-GM and CFU-M) and total CFU at 5 lowed by increased proliferation by 7 days of treatment.
days postirradiation (Fig. 4B). Trends were observed for Our experiments demonstrate that captopril administration
improved recovery of CFU-GEMM and BFU-E, but these beginning 1 hour or 24 hours after irradiation and
did not reach significance. At day 14 postirradiation, there continuing for 7 to 30 days increased survival. Therefore,
was a significant increase in the total CFU in mice treated captopril-induced radiation protection correlated with tran-
with captopril postirradiation compared with mice that sient quiescence (increased G ) of the ST-HSC population
0
receivedradiationalone(Fig.4C).Trendstowardincreased and prevention of stem cell pool exhaustion. However,
levels of CFU-GM and CFU-M progenitor cell activity when captopril was initiated 7 days before irradiation and
were also observed in mice that received captopril treat- continued either to the time of irradiation or for an addi-
ment postirradiation. tional 30 days postirradiation, a significant increase in
mortality was observed compared to untreated irradiated
Effectofcaptopriloncirculatinghematopoieticcellsand mice. In this case, radiation sensitization was correlated
marrow-derived HPC with increased cycling (increased G /M) of the ST-HSC
2
ResultsfrommaturebloodcellcountsandCFUassayssug- population at the time of radiation exposure.
gestedthatcaptoprilaffectedthesensitivityand/orrecovery HSC quiescence sustains long-term hematopoiesis by
ofhematopoieticcellsandtheirprecursors.Otherinvestiga- protectingtheHSCpool fromradiation-inducedinjury and
tors showed that Ang II directly regulates the growth and fromprematureexhaustionunderconditionsofhematopoi-
development of hematopoietic cells through angiotensin eticstress.Ontheotherhand,prematureentryofHSCinto
receptors on the surface of hematopoietic cells themselves the cell cycle following radiation exposure exhausts the
[26,27]. To investigate the effect of captopril on the HPC stemcell pool and leads to hematological failure [30]. The
cell-cycle profile, total Lin(cid:2) and Lin(cid:2) HPC (Lin(cid:2) Sca-1þ G /M phases of the cell cycle, as well as increased rate of
2
cKitþ [LSKþ]) femoral bone marrow cells were isolated cellcycling,areassociatedwithincreasedsensitivitytoradi-
fromnonirradiatedmiceafter2or7daysofcaptopriltreat- ation[5,7].ExaminationoftheresponseofHPCinnonirra-
ment and stained with fluorescein isothiocyanate(cid:2)labeled diated mice indicated that 2 days of captopril
Ki-67 and 7-aminoactinomycin-D. Whereas 72% of bone administration resulted in HPC transiently withdrawing
marrow Lin(cid:2) cells were active in the cell cycle in control from the cell cycle with increased percentages of cells in
mice, the Lin(cid:2) cells from mice that received 2 days of theG /G phases.However,inmicethatreceivedcaptopril
0 1
captopril treatment exhibited a significant decrease in the for 7 days, the HPC re-entered the cell cycle, resulting in
percentage of cells that were actively proliferating. This higher percentages of cells in the radiation-sensitive G /M
2
decrease was followed by a stronger G to G /M/S transi- phasesofthecycle.Therefore,exposureofthemorerapidly
1 2
tion after 7 days of captopril treatment (Fig. 5A). In cycling cells to radiation resulted in greater damage to the
contrast to what was observed for Lin(cid:2) cells, the majority cells. This was reflected by the significant decreases in
oftheLSKþremainedintheG phaseofthecellcycleafter CFU of multilineage progenitor cells observed at 5 days
0
T.A.Davisetal./ExperimentalHematology2010;38:270–281 277
Figure4. Effectofcaptoprilonnucleatedcellbonemarrowreconstitutioninirradiatedmice.Micewereuntreatedirradiated,6.0Gytotalbodyirradiation
(TBIcontrolgroup);pretreatedwithcaptopril(0.1mg/kg/day)for7consecutivedaysbeforeirradiation;orirradiatedandthengivencaptopril(0.1mg/kg/
day)for14consecutivedays.Atdays5and14postirradiation,femoralbonemarrowcellswerecollected.(A)Totalnumberofnucleatedleukocytescellsper
femurpostirradiation.Thenumberofassayablemultilineageandlineage-specificcolony-formingprogenitorcellsweredeterminedfrombonemarrowcells
collected at day 5 (B) and day 14 (C) postirradiation. Multipotential (colony-forming unit granulocyte-erythroid-macrophage-megakaryocyte [CFU-
GEMM]),myeloid(CFUgranulocytic-macrophage[CFU-GM],CFUmacrophage[CFU-M]),anderythroid(blast-forminguniterythroid([BFU-E])bone
marrowcolony-formingcellsweredetermined.Forallpanels,resultsrepresentmean6standarderrorofmeanofsixmice.*p!0.05,comparedwith
TBI-treated group. For comparison purposes, the bone marrow cellularity of untreated nonirradiated micewas 20.37 (cid:4) 106 nucleated leukocytes/femur
(datanotshown).
postirradiation,whichultimatelyresultedinincreasedhema- and increased susceptibility to radiation therapy. This may
topoietic failure. These findings suggest that HSC from haveledtoimpairedlong-termrepopulatingpotential,hema-
captopril-pretreated mice exhibit a higher activated state topoietic exhaustion, bone marrow failure, and decreased
that is associated with a loss of HSC cell-cycle quiescence survival.