Severe Ocular Abnormalities in C57BL/6 but Not in 129/Sv p53-Deficient Mice

85 Pages·2012·2.5 MB·English

R E P O R T S
Caspaselike Proteases Activated In Royal College of Surgeons (RCS) rats, photoreceptor de-
generation caused by impaired phagocytosis of rod outer
in Apoptotic Photoreceptors of
Royal College of Surgeons Rats postnatalday20(P20),1andalmostallphotoreceptorsdieout
byP60.Recently,thisretinaldegenerationhasbeenshownto
be caused by apoptotic cell death, based on the finding that
Naomichi Katai, Takanobu Kikuchi, cellsintheouternuclearlayer(ONL)arestainedbyTdT-dUTP
Hiroto Shibuki, Sachiko Kuroiwa, Jun Arai, terminal nick—end labeling (TUNEL) and that agarose gel
electrophoresis of DNA extracted from the retina shows a
Toru Kurokawa, and Nagahisa Yoshimura
typicalladderpattern.2
Apoptosis is a phenomenon underlying normal develop-
PURPOSE.Tostudytheroleofcaspase-likeproteases,espe- mentandmanypathologicconditionsandisachievedthrough
cially roles of more extensively characterized caspase-1 activationofacascadecalleda“deathprogram.”3,4Ingeneral,
and caspase-2, in apoptotic photoreceptor cell degenera- the apoptotic process is divided schematically into five steps:
tioninRoyalCollegeofSurgeons(RCS)rats. activation, propagation, commitment, execution, and, finally,
cell death.3Interleukin-1 (cid:98)–converting enzyme (ICE)/caspase
METHODS.BothRCSandSprague—Dawleyratswereused. family proteins, identified by their homology with the nema-
Cryosectionsoftheretinasatvariouspostnataltimeswere
todedeathgeneced-3,areexecutorsoftheapoptoticprogram
immunostained with antibodies against caspase-1 (inter- insomevertebratecells.4Althoughthefinalstepofapoptosis
leukin-1(cid:98)–converting enzyme, ICE) and caspase-2 is invariably cell death, various signals contribute to the final
(Nedd2/Ich-1). Double staining with TdT-dUTP terminal commonpathway,andintermediatestepsdifferdependingon
nick–end labeling (TUNEL), propidium iodide, and the the etiology of the apoptosis and cell type.4 If the apoptotic
antibodies was also performed. To evaluate the time process has not proceeded beyond the execution step, a cell
course of protein expression, western blot analysis was can be rescued in some instances by blocking expression of
carriedout.Thetemporalprofileofcaspase-likeprotease apoptosis-relatedgenesorbyoverexpressionofbcl-2.4
activitywasstudiedusingafluorogenictetrapeptidesub- Apoptosisplaysamajorroleinthepathogenesisofmanydiseasesinwhichphotoreceptorsdegenerate,suchasretinitis
strate, acetyl-tyrosyl-valyl-alanyl-aspartic acid (cid:97)(4-methyl- pigmentosa, retinal detachment, light injury, ischemic injury,
coumaryl-7-amide) (Ac-YVAD-MCA). Intravitreal injection andage-relatedmaculardegeneration.
ofacaspase-1inhibitor,acetyl-tyrosyl-valyl-alanyl-aspartic- 5,6Therefore,itisimpor-
aldehyde(Ac-YVAD-CHO),atpostnataldays21(P21)and tant to know which genes are specifically expressed during
apoptosis of photoreceptor cells. Herein, we will show that
P26 was performed to see if this caused a decrease in
members of the caspase family may play a critical role in the
apoptoticcellnumberatP28.
apoptosisofphotoreceptorcellsintheRCSrat.Inthepresent
RESULTS. TUNEL-positive photoreceptors of RCS rats study, we concentrated on caspase-1 and caspase-2 because
stained strongly with antibodies against caspase-1 and theseenzymesareknowntoplayanimportantroleinneuronal
caspase-2.Doublestainingstudiesrevealedthatcaspase-1 apoptosisofischemicbraininjuryandretinaldevelopmentand
andcaspase-2werecoexpressedinapoptoticcells.West- apoptosis of rat pheochromocytoma-derived cell line cells
ern blot analysis showed that active forms of caspase-1– (PC12).7–9
likeandcaspase-2–likeproteaseswereupregulatedatP28,
concurrent with the peak in TUNEL-positive cells. The
enzymaticactivityofcaspase-1–likeproteasewaselevated MATERIALS AND METHODS
in RCS rat retinas at P28, and the inhibitor of caspase-1
AnimalsThe care and maintenance of rats conformed to the ARVO
transientlyreducedthenumberoftheapoptoticphotore-
Statement for the Use of Animals in Ophthalmic and Vision
ceptors.
Research. RCS (Jcl-rdy/rdy) rats were obtained from Japan
CONCLUSIONS.Activationofcaspase-likeproteasesplaysan CLEA, Tokyo, Japan, and Sprague—Dawley (SD) rats from a
important role in photoreceptor apoptosis of RCS rats.
local breeder. Rats were maintained under 12-hour light/12-
(InvestOphthalmolVisSci.1999;40:1802–1807)
hourdarkconditions.RCSandSDratsof14,21,25,28,33,35,
45, and 56 days of age were used in this study.
From the Department of Ophthalmology, Shinshu University
SchoolofMedicine,Matsumoto,Japan. Antibodies
Supported by Grant-in-Aid 10470567 (NY) from the Ministry of
Education,Culture,SportsandScienceoftheJapaneseGovernment. Antibodies used in this study were obtained from various
Submitted for publication June 24, 1998; revised October 28, sources:Goatanti–caspase-1(ICE)polyclonalantibody(M-19)
1998;acceptedDecember17,1998. andgoatanti–caspase-2(Nedd2/Ich-1)polyclonalantibody(N-
Proprietaryinterestcategory:N.
19) were purchased from Santa Cruz Biotechnology (Santa
Reprint requests: Nagahisa Yoshimura, Department of Ophthal-
mology,ShinshuUniversitySchoolofMedicine,Matsumoto390-8621, Cruz, CA); fluorescein isothiocyanate (FITC)– and rhodamine-
Japan.
InvestigativeOphthalmology&VisualScience,July1999,Vol.40,No.8
1802 Copyright©AssociationforResearchinVisionandOphthalmology
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IOVS,July1999,Vol.40,No.8 Reports 1803
mark); and FITC- and rhodamine-conjugated rabbit anti-goat mM phenylmethylsulfonyl fluoride, 10 (cid:109)g/ml pepstatin, 20
IgGfromJacksonImmunoResearchLaboratories(WestGrove, (cid:109)g/ml leupeptin, and 10 (cid:109)g/ml aprotinin. Sodium dodecyl
PA).Thesecaspaseantibodiesrecognizebothproenzymeand sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was
active forms of protease. Anti-phosphotyrosine antibody from carried out as described by Laemmli.11 A 50-(cid:109)g sample of
UpstateBiotechnologywasusedtodistinguishphotoreceptors protein was loaded on each lane, electrophoresed with 10%
frommicroglialcellsandperipheralmacrophages. acrylamide gel, and then transferred to nitrocellulose mem-
branes. After being rinsed with Tris-buffered saline (pH 7.4)
TUNEL and Propidium Iodide Staining
containing 0.25% Tween-20 (TBST), the membrane was incu-
DNA nick end-labeling was performed according to a slightly bated with the primary antibodies (0.5 (cid:109)g/ml) in TBST for 1
modified method of Gavrieli et al.10 After rats were anesthe- hour at room temperature. After three washings, the mem-
tized with an intraperitoneal injection of pentobarbital (50 brane was incubated for 1 hour at room temperature with
mg/kg), they were perfused transcardially with 100 ml of horseradish peroxidase–conjugated secondary antibody (Am-
normalsalinesupplementedwith1U/mlheparinfollowedby ersham, Buckinghamshire, UK) diluted 1:500 in TBST. The
200mlof4%paraformaldehydein0.1Mphosphatebuffer(pH membrane was washed again and developed with the chemi-
7.4). The eyes were enucleated and placed in 4% paraformal- luminescenceECLwesternblottingsystem(Amersham).
dehydefor24hoursat4oC.Theywererinsedwithphosphate-
buffered saline (PBS) and then transferred to 10%, 15%, and Intravitreal Administration of Caspase-1 Inhibitor
20% sucrose in 0.1 M PBS for 3 hours at room temperature. A specific inhibitor of caspase-1, acetyl-tyrosyl-valyl-alanyl-as-
Sampleswerethensnap-frozeninTissue–Tek(MilesLaborato- partate-aldehyde(Ac-YVAD-CHO),wasobtainedfromthePep-
ries, Elkhart, IN) on dry ice in hexane and stored at (cid:50)80oC. tideInstitute(Osaka,Japan).A20-mMsolutionoftheAc-YVAD
Cryostated sections (10–15 (cid:109)m) were thaw-mounted onto in dimethyl sulfoxide was prepared, and 1 (cid:109)l of the solution
glassslidescoatedwithpoly-L-lysineandair-driedfor2hoursat wasinjectedintothevitreouscavityoftheRCSratsatP21and
roomtemperature.Aftercryosectionswererinsedthreetimes P26bymeansofa30-gaugeneedle.Asacontrol,1(cid:109)ldimethyl
in 0.02 M PBS (pH 7.4), sections were incubated with biotin- sulfoxide (vehicle) was injected in the same way. At P28, the
ylatedterminaldUTPtransferaseandbiotinylateddUTPinTdT eyeswereenucleated,andtheTUNELstainingwasperformed
buffer(30mMTris,pH7.2,140mMsodiumcacodylate,and1 asdescribedabove.
mM cobalt chloride) for 60 minutes at 37oC with terminal
dUTPinthemoisturechamber.Afterrinsing,thesectionswere Assay of Caspase Activity
reactedwithavidin–FITCandexaminedwithaZeissscanning
Caspase-1–like protease activity was assayed as described by
laser confocal microscope (LSM 410 inverted Laser Micro-
Nicholson et al.12 with slight modification.13 Briefly, resected
scope; Zeiss, Oberkochen, Germany). Positive controls were
retinas were homogenized in 10 mM Hepes–KOH, pH 7.2, 2
generatedusingDNaseIinTdTbuffer(1(cid:109)g/ml)beforeincu-
mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol, 1 mM phenyl-
bation with terminal transferase and biotinylated nucleotides.
methylsulfonylfluoride,10(cid:109)g/mlpepstatin,20(cid:109)g/mlleupep-
Propidium iodide (PI) staining was performed by incubating
tin,and10(cid:109)g/mlaprotinin.Homogenateswerecentrifugedat
thesectionswith50(cid:109)lPI(20(cid:109)g/ml)for10minutesatroom
15,000g for 30 minutes, after which the supernatants were
temperature.TUNELstainingofcells,compactednuclearstain-
transferredtonewEppendorftubes.Aliquotsofextracts(300
ing, nuclear fragmentation, and loss of nuclear staining by PI
(cid:109)g protein in 100 (cid:109)l extraction buffer) were preincubated at
wereconsideredtobesignsofapoptosis.
37°C for 30 minutes and then mixed with 5 (cid:109)l of 10 mM
Immunohistochemistry tetrapeptidesubstrate,acetyl-tyrosyl-valyl-alanyl-asparticacid(cid:97)
(4-methyl-coumaryl-7-amide) (Ac-YVAD-MCA; (Peptide Insti-
After rinsing with PBS, cryosections were incubated with 2%
tute).Freeaminomethylcoumarin(AMC)accumulation,which
normalgoatorrabbitserumfor60minutesatroomtempera-
resulted from cleavage of the aspartate-AMC bond, was moni-
ture.Incubationwiththeprimaryantibody(anti–caspase-1,10
tored in each sample at 37°C over 180 minutes using a spec-
(cid:109)g/ml, and anti–caspase-2, 10 (cid:109)g/ml) was carried out in a
trophotometer(UltraspecIII;Pharmacia,Cambridge,England).
moisture chamber at 4oC overnight. FITC- or rhodamine-con-
The absorbance of the each sample at 370 nm was plotted
jugated secondary antibody was reacted at room temperature
againsttime.Linearregressionanalysisofthevelocityofeach
for2hours.
curve yielded the activity for each sample. Data were ex-
Quantitative Analysis of TUNEL and pressed as a percentage of the caspase-1–like activities in the
Immunohistochemistry samplescomparedtothoseintheP28SDratretina.
At each point of the time course study for TUNEL and immu-
Statistical Analysis
nohistochemical staining, the number of TUNEL-positive- and
immunostainedcellsintheONLwasdeterminedin5meridian The data were analyzed statistically by one-way ANOVA fol-
sections through the optic nerve. The numbers of cells were lowed by Fisher’s post-hoc test. Probability values less than
countedin0.4-mmlengthsofthesection1mmfromtheoptic 0.05weredeterminedtobestatisticallysignificant.
nerveheadonthesuperiorandinferiorhemisphereusingthe
Zeiss confocal microscope with the “measure” function. Re-
sultsareexpressedasmean(cid:54)SEM. RESULTS
Analysis of Western Blots TUNEL Staining
Samples (n (cid:53) 5 at each time) were homogenized in buffer InbothRCSandSDrats,TUNEL-positivecellswereobservedin
containing Tris–HCl (pH 8.0), 250 mM NaCl, 0.5% NP-40, 0.1 theONLatP14.InSDrats,thesecellswerenolongerobservedat
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1804 Reports IOVS,July1999,Vol.40,No.8
FIGURE1. (A)ThetimecourseofTUNELstainingin
theONLoftheRCSrat.Theimagesrepresentdigital
overlay of a black-and-white image using Nomarski
differential interference contrast and a green color
image of TUNEL staining using a confocal micro-
scope.(B)ThenumberofTUNEL-positivenucleiin
the ONL of the RCS rats. TUNEL-stained cells are
countedin0.4-mmcryosectionsintheONLofeach
retina using a confocal microscope. Data are ex-
pressedasthemean(cid:54)SEM(n(cid:53)4).
P21.Incontrast,TUNEL-positivecellsincreasedinnumberinthe densednuclei.IncellswithweakornoPIstaining,caspase-1–
ONLofRCSratsatP21toP45,withapeakatP28(Figs.1A,1B). like and caspase-2–like immunoreactivities were observed in
At P28, the number of TUNEL-positive cells was approximately thenucleusandcytoplasm.Immunopositivecellspeciescould
250/0.4-mmsection,andaboutonethirdofthetotalcellnumber notbeidentifiedbecauseapoptoticcellshadlosttheiroriginal
intheONLatP28wasstainedbyTUNEL(Fig.1B). appearance.However,weconcludedthattheimmunostained
cellswereapoptoticphotoreceptorsbecauseoftheirlocation
Immunohistochemistry
in the ONL and because they were not immunolabeled with
SpecificstainingofcellsintheONLofRCSratswasobtained anti-phosphotyrosineantibody(graphicdatanotshown).
usingantibodiesagainstcaspase-1andcaspase-2(Figs.2A,2B).
TUNEL-Positive and Immunostained Cells
In contrast, no specific staining was detected by any of the
antibodies in the ONL of P28 SD rat retinas (graphic data not InRCSrats,quantitativeanalysesshowedthatthenumbersof
shown). cells stained with antibodies against caspase-1 and caspase-2
Diffusecytoplasmicstainingwithanti–caspase-1andanti– were very similar during the follow-up period (Fig. 2C). Also,
caspase-2antibodieswasseeninapoptoticcellsthathadcon- the time course of TUNEL-positive cells showed a similar pat-
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IOVS,July1999,Vol.40,No.8 Reports 1805
FIGURE 2. (A, B) Confocal micro-
scopicimagesofTUNELandimmu-
nostainingofRCSratretina.TUNEL
staining(a)andimmunostaining(b)
with rhodamine-conjugated second-
aryantibodiestoanti–caspase-1(A)
andanti–caspase-2(B)antibody,No-
marski differential interference con-
trast (c), and digital overlay (d) of
TUNELandimmunostaining.TUNEL-
positive cells were colabeled with
antibodiesagainstcaspase-1(A)and
caspase-2(B)intheONLofP28RCS
rats.(C)Timecourseofimmunopo-
sitive cells in the ONL of RCS rats.
The temporal profile of immuno-
stainingwassimilartothatofTUNEL
staining. Caspase-1–positive cells
were seen more frequently than
were caspase-2–positive cells. Data
arerepresentedasfindingsper4-mm
tissue section and are expressed as
mean(cid:54)SEM(n(cid:53)4).Scalebar,50
(cid:109)m.
ternofimmunostaining.Thenumbersofimmunostainedcells Immunoblot Analysis of Caspase-1– and Caspase-
were almost equal to that of TUNEL-positive cells at P14 and 2–Like Proteases
P21 (less than 6 cells/0.4-mm tissue section), but there were
AsshowninFigure3A,anti–caspase-1antibodydepictedboth
approximately10timesmoreTUNEL-positivecellsthanimmu-
37.5-kDa and 20-kDa bands by western blot analysis. The ap-
nostained cells at P28 (243.3 (cid:54) 8.0 TUNEL-positive cells/
parent molecular weight of the bands agrees with that of
0.4-mmtissuesection,18.2(cid:54)0.4caspase-1–positivecellsand
caspase-1–like protease. Figure 3B shows that caspase-2–like
13.7(cid:54)0.4caspase-2–positivecells).
protease is expressed in RCS rat retinas, based on the finding
Double staining studies to identify TUNEL-positive cells
that anti–caspase-2 antibody blotted at both 40.5- and 33-kDa
and immunostained cells using anti–caspase-1 and anti–
bands. The active form of caspase-1 and caspase-2 consists of
caspase-2 antibodies showed that these signals were colocal-
long and short fragments that are derived from proteolytic
ized in the same cell (Figs. 2A, 2B). Caspase-1 and caspase-2
processing of the proenzyme during apoptosis. Antibodies
were also double-stained in the same cell (graphic data not
used in this study reacted with the active form of the 20-kDa
shown). However, all the anti–caspase-1–positive cells were subunitofcaspase-1andthe33-kDasubunitofcaspase-2.The
notalwaysstainedwithanti–caspase-2antibodies.Theresults level of expression of the 37.5 kDa caspase-1 subunit was
of double staining with the antibodies and TUNEL staining constant,butthatofthe20-kDasubunit,theactiveform,was
showedthatexpressionofcaspase-1andcaspase-2wascorre- upregulatedatP21,P28,andP35RCSrat.Ontheotherhand,
latedwithphotoreceptorapoptosisinRCSrats.Anti-phospho- expressionofthe40.5-kDaproenzymewasconstant,whereas
tyrosine antibody–labeled cells did not show coexpression of thatofthe33-kDaactiveformofcaspase-2wasupregulatedat
caspase-1orcaspase-2(graphicdatanotshown). P14,P21,P28,andP35inRCSratretinas.
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1806 Reports IOVS,July1999,Vol.40,No.8
FIGURE 5. TUNEL-positive cells in the ONL of P28 RCS rats after
intravitrealinjectionofthecaspase-1inhibitorAc-YVAD-CHO.Dataare
expressed as mean (cid:54) SEM (n (cid:53) 4). Photoreceptor apoptosis was
transientlyblockedbythecaspase-1inhibitor.*P(cid:44)0.01.
FIGURE3. Westernblotanalysisofcaspase-1–like(A)andcaspase-2–
like(B)proteinsintheRCSrat.(A)Bandsof20and37.5kDawere
depictedwiththeanti–caspase-1antibody.Expressionofthe37.5-kDa likeproteaseactivitywaselevatedintheextractfromP28RCS
subunit was at a constant level, but that of the 20-kDa subunit was ratretinas.ThelevelsoftheactivityweretwofoldthoseofP14
upregulatedatP28andP35.(B)Anti–caspase-2antibodyshowed40.5- rats at P21 and sevenfold at P28. The time course of caspase
and 33-kDa bands. Both subunits were upregulated at P28. Data are activitychangeswassimilartothoseofimmunohistochemical
representativeoffiveseparateexperimentswithsimilarresults.
analysis and protein expression level of caspase-1 active form
asshowninwesternblotanalysis.
Assay of Caspase-1–Like Protease Enzymatic
Effect of the Caspase-1 Inhibitor
Activity
Figure 5 indicates that the number of TUNEL-positive cells in
Activity of caspase-1–like protease was assayed using the spe-
ONLwassignificantlyreducedinP28RCSratretinasinjected
cific tetrapeptide substrate. As shown in Figure 4, caspase-1–
withtheinhibitorAc-YVAD-CHOatP26.However,thenumber
of TUNEL-positive cells of P28 rat retinas injected with the
tetrapeptide at P21 was not decreased. These results indicate
that the caspase-1 inhibitor can suppress, at least transiently,
apoptosisofphotoreceptorcells.
DISCUSSION
In this study, we demonstrate that caspase-1– and caspase-2–
likeproteasesplayanimportantroleinphotoreceptorapopto-
sis during retinal degeneration in the RCS rat. Activation of
caspase-1–like protease contributes to photoreceptor apopto-
sisintheRCSrat.
Caspase-1 was first identified as the mammalian homo-
logue of the ced-3 gene, initially identified in Caenorhabditis
elegans. Caspase-1 is known to promote apoptosis during de-
velopment of the nematode, and, currently, 10 Ced-3–related
cysteine proteases, termed caspase, have been cloned.3,4
These family members participate in one of two distinct sig-
naling pathways: activation of proinflammatory cytokines and
FIGURE4. Caspase-1–likeenzymaticactivitiesintheretinalextractsof
activation of apoptotic cell death.4 All the known caspase
RCSrats.ProteaseactivityisexpressedasapercentageofthatinP28
SD rat retina. Data are expressed as the mean (cid:54) SEM (n (cid:53) 4). The family proteases are synthesized as inactive proenzymes,
enzymaticactivitiesatP28aresignificantlyelevatedrelativetothoseof which require cleavage to liberate one large and one small
SDatP28.*P(cid:44)0.01. subunittoformtheactiveenzyme.4Activationofonecaspase
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IOVS,July1999,Vol.40,No.8 Reports 1807
canleadtocleavageandactivationofanothermoleculeofthe sections.Athirdpossibilityisthatnumerousothermechanisms
samecaspase,anothercaspase,orboth,leadingtoanamplified maybeinvolvedintheapoptoticprocessinRCSrats,andwe
apoptotic cascade.4 Indeed, in the Fas-mediated apoptosis of maybeawareofonlyasmallportionofthisapoptoticprocess.
lymphoidcells,activationofcaspase-3viaspecificcleavageof Forexample,caspasesotherthancaspase-1andcaspase-2may
the proenzyme by caspase-1 has been demonstrated.4 A num- playamoreimportantroleintheapoptoticprocessoftheRCS
ber of experimental studies suggest that activation of the rat retina. Finally, it is likely that the persistence of apoptotic
caspasefamilyplaysacriticalroleintheexecutionofapoptotic nucleiforlongperiodsreflectsafailureofphagocytosis.
events.3,7–9 Caspase-1 was implicated in death caused by su- Toourknowledge,thisisthefirstreportofacorrelation
peroxide dismutase downregulation in PC12 cells but not in between photoreceptor apoptosis and the caspase family. Al-
withdrawal of trophic factor support.9 Conversely, the anti- thoughthisstudysuggestsanimportantroleforcaspase-1and
sense constructed to downregulate caspase-2 in PC12 cells caspase-2intheprocessofphotoreceptorapoptosisintheRCS
inhibitedcelldeathbywithdrawaloftrophicsupportbutnot ratretina,furtherstudiesarenecessarytoclarifythemolecular
oxidative stress.7 The activation of distinct caspases in the mechanismunderlyingretinaldegenerationintheseanimals.
samecellsthuscanpromoteapoptosisinitiatedbythevarious
stimuli.7
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VEGF Increases Retinal Vascular nopathy in nonhuman primates, including retinal ischemia.4
The cellular and molecular mechanisms underlying this phe-
ICAM-1 Expression In Vivo
nomenonhavenotbeendefined.
Ming Lu,1,2,5 Victor L. Perez,3,5 Naili Ma,3,5 Fluoresceinangiographicstudiesinhumanshavedemon-
Kazuaki Miyamoto,1,2 Hai–Bing Peng,4 stratedthatsomediabeticretinalischemiaisreversible.5How-
ever, in most eyes a large proportion of the diabetic retinal
James K. Liao,4 and Anthony P. Adamis1,2
ischemia that develops is irreversible and results from the
formationofacellularcapillaries.6Diabeticretinalleukostasis7
PURPOSE. Intraocular injections of vascular endothelial may be operative in the development of both types of retinal
growthfactor(VEGF),apeptideimplicatedinthepatho- ischemia. Increased numbers of monocytes and granulocytes
genesis of diabetic retinopathy, can induce retinal isch- havebeendemonstratedtooccupythelumensofretinalcap-
emia.Diabeticretinalischemiamaybecaused,inpart,by illaries and postcapillary venules in diabetic rats, with some
the adhesion of leukocytes to the retinal vasculature. In leukocyteslyingadjacenttodyingendothelialcells.8Increased
thisstudy,theabilityofVEGFtoincreasetheexpression numbers of granulocytes have also been demonstrated in the
of intercellular adhesion molecule-1 (ICAM-1) and other retinasofdiabetichumans.9Onepotentialmechanismmediat-
adhesion molecules in capillary endothelium and the ret- ingthisphenomenonistheincreasedadherenceofleukocytes
inalvasculaturewasexamined. tothevascularendothelium.Intercellularadhesionmolecule-1
METHODS. The expression of ICAM-1, vascular cell adhe- (ICAM-1)mediatestheadhesionofneutrophilsandmonocytes
sion molecule-1 (VCAM-1), E-selectin, and P-selectin on to vascular endothelium, and increased ICAM-1 levels have
human brain capillary endothelial cell monolayers ex- been found in the retinal vasculature of human diabetics.9 In
posed to VEGF was quantitated by immunoassay. The addition, human diabetic neutrophils have been shown to be
effectofVEGFonretinalvascularICAM-1expressionwas morepronetoupregulateICAM-1ligandCD18.10
determinedinICAM-1immunofluorescencestudiesofret- Becauseoftheseobservations,studieswereundertakento
inalflat-mountsandinRNaseprotectionassays. characterizetheeffectofVEGFontheexpressionofendothe-
lialcelladhesionmoleculesinvitroandinretinainvivo.
RESULTS.VEGFincreasedcapillaryendothelialcellICAM-1
levelsinadose-andtime-dependentmanner(6–24hours,
plateauafter6hours;EC ,25ng/ml).VEGFfailedtoalter
50 METHODS
E-selectin, P-selectin, or VCAM-1 levels under the condi-
tionstested.Intravitrealinjectionsofpathophysiologically
Cell Culture
relevantconcentrationsofVEGFincreasedICAM-1protein
andmRNAlevelsintheretinalvasculature. Human brain capillary endothelial cells (HBCEC) were ob-
tained from Cell Systems Corporation (Kirkland, WA) and
CONCLUSIONS. VEGF increases retinal vascular ICAM-1 ex-
maintained in medium 199 (GIBCO BRL, Gaithersburg, MD),
pression.VEGF-inducedincreasesinICAM-1maypromote
containing HEPES (25 mM), heparin (1%), endothelial cell
retinal leukostasis in diabetic eyes. (Invest Ophthalmol
growthfactor(50(cid:109)g/ml),L-glutamine(2mM),100U/mlpen-
VisSci.1999;40:1808–1812)
icillin, 100 mg/ml streptomycin, and 15% fetal bovine serum
(HyCloneLaboratories,Logan,UT),accordingtothemanufac-
Vascularendothelialgrowthfactor(VEGF)isamajormedi- turer’s instructions. For enzyme immunoassay (EIA), HBCECs
ator of retinal ischemia–associated ocular neovasculariza- weregrownontissueculture–treatedplasticmicrotiter96-well
tion. Intraocular VEGF levels have been temporally and spa- plates, coated with 0.1% (wt/vol) gelatin (Difco, Detroit, MI),
tially correlated with neovascularization,1 and the specific andallowedtoreachconfluence.
inhibition of VEGF in animal models has been shown to sup-
presstheneovascularizationoftheiris2andtheretina.3More Adhesion Molecule Quantitation via EIA
recently, exogenous VEGF has also shown to induce many of Endothelial cell adhesion molecule levels were measured by
theangiographicandhistopathologicfeaturesofdiabeticreti- EIAinHBCECs.HBCECsweregrowntoconfluencein96-well
plates and treated with serum-free medium with or without
variousconcentrationsofrecombinanthumanVEGF (agift
165
Fromthe1LaboratoryforSurgicalResearch,Children’sHospital, of Napoleone Ferrara, Genentech, South San Francisco, CA;
Boston, Massachusetts; the 2Department of Ophthalmology, Harvard endotoxin(cid:44)0.03EU/ml)orrecombinanthumantumornecro-
Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massa- sis factor-(cid:97)(TNF-(cid:97); R&D Systems, Minneapolis MN; positive
chusetts;the3SchepensEyeResearchInstitute,Boston,Massachusetts;
and4BrighamandWomen’sHospital,Boston,Massachusetts. control)for24hours.CellsurfaceEIAswerecompletedusing
5These authors contributed equally to the work presented here mouse anti-human monoclonal antibodies against ICAM-1 (Ab
andshouldthereforeberegardedasequivalentseniorauthors. HU 5/3), VCAM-1–1 (Ab E1/6), and E-selectin (Ab H18/7).11
SupportedbytheNationalEyeInstitute(APA),theMassachusetts
TheEIAswerecarriedoutbyincubatingmonolayersfirstwith
Lions,andtheRobertaW.SiegelFund(APA).
saturating concentrations of specific monoclonal antibodies
Presented in part at the 1997 and 1998 annual meetings of the
AssociationforResearchinVisionandOphthalmology,Ft.Lauderdale, againstthetargetmolecule,followedbybiotinylatedgoatanti-
Florida,May1997andMay1998. mouseIgG,andfinallywithstreptavidin-alkalinephosphatase.
SubmittedforpublicationJune30,1998;revisedJanuary8,1999; The surface expression of each protein was quantified spec-
acceptedFebruary16,1999.
trophotometrically, reading the optical density of the wells
Proprietaryinterestcategory:N.
Reprintrequests:AnthonyP.Adamis,MassachusettsEyeandEar (410nm)15to60minutesaftertheadditionofachromogen
Infirmary,243CharlesStreet,Boston,MA02114. (p-nitrophenylphosphate),asdescribedpreviously.11Thedata
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IOVS,July1999,Vol.40,No.8 Reports 1809
werenormalizedagainstproteinconcentrationsandexpressed
asmean(cid:54)SEM.
ICAM-1 Immunofluorescence
AnimalswerecaredforinaccordancewiththeARVOResolu-
tionontheUseofAnimalsinOphthalmicandVisionResearch..
The experiments were approved by the Schepens Eye Re-
search Institute Animal Care and Use Committee. Normal
C57B/6 mice (male, 6 to 8 weeks old) were examined for
retinalICAM-1expressionafterintravitrealinjectionsofrecom-
binant murine VEGF (R&D Systems, Minneapolis, MN) or
164
vehiclealone(n(cid:53)6;endotoxin(cid:44)0.1EU/mg).Afteranesthe-
sia with a mixture of ketamine (Ketalar; Parke–Davis, Morris
Plains,NJ;[150mg/kg])andxylazine(Rompun;Harver–Lock-
hart,MorrisPlains,NJ;[60mg/kg]),2(cid:109)lconcentratedVEGFor
phosphate-buffered saline (PBS) solvent was injected slowly
into the vitreous cavity to attain a final concentration of 100
ng/mlVEGF.After24hours,theanimalswerekilledbycervical
dislocation,andtheeyeswereenucleatedandimmersion-fixed
in0.5%(wt/vol)paraformaldehydein0.1MPBS(pH7.4)for30
minutes.Theintactretinasweredissectedoutunderasurgical
microscopeaspreviouslydescribed12andfurtherfixedin70%
ethanolatroomtemperatureforanadditional30minutes.The
retinaswerewashedin0.1MPBS(pH7.4)with0.1%(vol/vol)
TritonX-100for30minutesandthenincubatedwithbiotinyl-
atedanti-mouseICAM-1(1:50dilutedin0.1MPBScontaining
1% [wt/vol] bovine serum albumin, BSA) overnight at 4oC.
After washing at room temperature with PBS containing 1%
BSA, the retinas were further incubated with streptavidin–
fluoresceinisothiocyanate(1:100dilutedinPBScontaining1%
BSA; Amersham, Arlington Heights, IL) for 2 hours at room
temperature. An isotype-matched IgG was used as a negative
control. The retinas were co-stained with 2 ng/ml ethidium
bromide for 10 seconds and mounted. Endothelial cell nuclei
werecountedperunitlengthofvessel.
RNase Protection Assay for ICAM-1
TheexperimentswereapprovedbyChildren’sHospitalAnimal
CareandUseCommittee.MaleSprague–Dawleyratsweighing
200 to 250 g were anesthetized with 0.1 mg/kg sodium amo- FIGURE1. Time(A)anddose(B)responsesofsurfaceICAM-1levels
in HBCECs after exposure to serum-free medium with and without
barbital. Pupils were dilated with 0.5% tropicamide and 2.5%
VEGF . Time course experiment was performed using 50 ng/ml
phenylephrinehydrochloride(Alcon,Humancao,PuertoRico). 165
VEGF .DoseresponseofICAM-1(B)wasperformedaftera24-hour
MurineVEGF165orhumanTNF-(cid:97),inatotalvolumeof5(cid:109)lPBS, expos1u6r5etoserum-freemediumwithandwithoutVEGF165.*P(cid:44)0.05
wasinjectedintotherightvitreousatasite1mmposteriorto vscontrol,n(cid:53)8.Dataareexpressedaspercentageovercontrol(no
the limbus using a Hamilton syringe with a 30-gauge needle. VEGF treatment) optical density, normalized against protein concen-
ThefinalconcentrationsofVEGFandTNF-(cid:97)werecalculatedto trationineachwell.
be 100 and 10 ng/ml, respectively. The contralateral control
eyes received 5-(cid:109)l PBS injections. The eyes were enucleated
2.5hourslater.Theratswereeuthanatizedwithananesthetic probe of 225 nucleotides (nt). This probe protects a 166-nt
overdosefollowedbyCO incubation.Allinjectionsweredone fragment of ICAM-1. All samples were simultaneously hybrid-
2
underdirectobservationusingasurgicalmicroscope.Anyeyes izedwithan18Sriboprobe(Ambion,Austin,TX)tonormalize
that had damage to the lens or retina were not used for forvariationsinloadingandrecoveryofRNA.Full-lengthpro-
analyses.Theretinasweregentlydissectedfreeandcutatthe tectionofthisproberesultsinan80-ntfragment.Theassaywas
disc.ThetissuewasplacedinanEppendorftube,snap-frozen performedaspreviouslydescribed.13Tenmicrogramsoftotal
in liquid nitrogen, and stored at (cid:50)80oC. The retinas were cellularRNAwashybridizedwith32P-labeledantisenseICAM-1
homogenizedin1mlRNAzol(BiotecxLaboratories,Houston, and18Sriboprobes(200,000cpmofeach)overnightat42oCin
TX)at4oCandpreparedforRNaseprotectionassays. 30(cid:109)lhybridizationbuffer.HybridizedRNAwasdigestedwith
The ICAM-1 riboprobe was produced by subcloning the nucleaseP1(20(cid:109)g/ml)andRNaseT1(2(cid:109)g/ml)for1hourat
coding sequence of the rat ICAM-1 cDNA into the EcoRI– 25oCin300(cid:109)ldigestionbuffer.Digestionswereterminatedby
BamHIsiteofthepBluescriptIIKSvector.TranscriptionbyT7 theadditionof20(cid:109)lof10%sodiumdodecylsulfateand50(cid:109)g
RNA polymerase after linearization by EcoRI resulted in a proteinaseKfor15minutesat37oC.Afterphenol/chloroform
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1810 Reports IOVS,July1999,Vol.40,No.8
extraction and ethanol precipitation, the protected fragments
wereresolvedon6%polyacrylamide,7Mureagelsandquan-
tifiedwithaPhosphorImager(MolecularDynamics,Sunnyvale,
CA).
Statistics
Significance was calculated using the paired Student’s t-test.
P(cid:44)0.05wasdeemedsignificant.
RESULTS
VEGFincreasedtheexpressionofICAM-1onHBCECinatime-
and dose-dependent manner (6–24 hours, plateau after 6
hours; EC , 25 ng/ml) (Figs. 1A, 1B). Expression levels of
50
surface VCAM-1 (Fig. 2A), E-selectin (Fig. 2B), and P-selectin
(Fig. 2C) were unaffected by incubation with VEGF for 6
165
hours (data not shown) or 24 hours. Incubation of HBCEC
monolayerswithTNF-(cid:97)(positivecontrol)producedsignificant
increases in VCAM-1 (2.4 (cid:54) 0.1–fold, n (cid:53) 8, P (cid:44) 0.05 versus
control), E-selectin (17.5 (cid:54) 1.3–fold, n (cid:53) 8, P (cid:44) 0.05 versus
control),P-selectin(10.7(cid:54)1.2–fold,n(cid:53)8,P(cid:44)0.01,datanot
shown), and ICAM-1 (2.5 (cid:54) 0.2–fold, n (cid:53) 8, P (cid:44) 0.05 versus
control,datanotshown).
Compared to the PBS-injected eyes, the VEGF-injected
eyes had a marked homogeneous upregulation of ICAM-1 im-
munoreactivity, particularly in the mid-peripheral retina and
the postcapillary venules. There was also a moderate upregu-
lation in the arterioles and capillary beds (Fig. 3). Because
VEGF is an endothelial cell mitogen, we examined whether
VEGF increased endothelial cell numbers, possibly confound-
ing the increases in ICAM-1 expression. Twenty-four hours of
VEGF treatment did not significantly increase the number of
endothelial cell nuclei per fixed segment length in the retinal
vasculature(datanotshown).
To examine whether VEGF increases ICAM-1 expression
at the transcript level, RNase protection assays of rat retinal
RNA were performed after intravitreal injections of VEGF or
PBS buffer. Compared to the PBS-injected eyes, the VEGF-
injectedeyeshada2.1(cid:54)0.4–fold(n(cid:53)4,P(cid:44)0.05)upregu-
lation of ICAM-1 mRNA (Fig. 4). As a positive control, TNF-(cid:97)
induced a 3.1 (cid:54) 0.8–fold increase of ICAM-1 mRNA (n (cid:53) 5,
P(cid:44)0.05).
DISCUSSION
ThesedatademonstratethatVEGFcanincreaseICAM-1expres-
sion on capillary endothelial cells in vitro and in the retinal
vasculature in vivo. The concentration of VEGF required to
increaseICAM-1invitro(EC ,25ng/ml)iscomparabletothat
50
measured in the vitreous of eyes with retinal ischemia and
neovascularization.1 The VEGF-induced effects appear to be
specificforICAM-1undertheconditionstested.Thesedataare
consistent with those of Melder and associates14 showing an
increaseinbioactiveICAM-1inangiogenictumorendothelium
in response to VEGF. However, our data differ regarding the
VEGF-induced upregulation of VCAM-1, because it was not FIGURE2. ExpressionofVCAM-1(A),E-selectin(B),andP-selectin(C)
observedinoursystem.Onereasonforthesedivergentresults in HBCECs after 24-hour exposure to serum-free medium with and
maybethedifferenttypesandstrainsofendothelialcellsused withoutVEGF orTNF-(cid:97).*P(cid:44)0.05vscontrol,n(cid:53)8.
165
inourstudies.
In addition to VEGF, other pathophysiologically relevant
stimulimayincreaseICAM-1inthediabeticretinalvasculature.
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IOVS,July1999,Vol.40,No.8 Reports 1811
FIGURE3. ICAM-1immunofluorescencestudyofC57B/6mouseretina24hoursafterexposureto100
ng/ml murine VEGF . Ethidium bromide staining identifies cell nuclei. Compared to the PBS-injected
164
eyes(A),theVEGF-injectedeyes(B)hadamarkedupregulationofICAM-1(1)intheretinalpostcapillary
venulesandveinsandamoderateupregulationinthearteriolesandcapillarybed.Twenty-fourhoursof
VEGF treatment did not significantly increase the number of endothelial cell nuclei in the retinal
vasculature(2).ICAM-1(1)andethidiumbromide(2)stainingareshownseparatelyandsuperimposed(3).
(Magnification,(cid:51)25.)
Factors relevant to diabetes, such as the cytokine TNF-(cid:97),15 retina. In the brain, ICAM-1 mediates leukocyte adhesion to
havebeenshowntoincreaseICAM-1inothersystems.TNF-(cid:97) postcapillaryvenulesandisassociatedwithcapillaryocclusion
levels are increased in the serum of diabetics,16 and as con- andvascularendothelialcelldamage.17
firmedbyourdata,TNF-(cid:97)isapotentinducerofendothelialcell Retinal VEGF levels appear to be elevated early in diabe-
ICAM-1expression.Asinothertissues,theinductionofICAM-1 tes, before there is any histopathologic evidence of retinal
may be causal for the leukostasis observed in the diabetic ischemia.18 This makes it more likely that endogenous VEGF
servestoincreaseretinalvascularICAM-1invivo.Wehypoth-
esize that VEGF, in whole or in part, increases retinal ICAM-1
expressionandthatICAM-1mediatesthebindingofleukocytes
tothevasculature,accountingfortheearlyreversiblephaseof
retinal ischemia. With time, the retinal leukostasis becomes
chronic and widespread, hastening the death of endothelial
cellsandpericytes.Withthedevelopmentofmorewidespread
ischemia, retinal VEGF levels are further upregulated, trigger-
inggreaterischemiaandultimatelyneovascularization.
This hypothetical sequence of events remains to be
proven. Proof requires the specific inhibition of VEGF and
ICAM-1 in the diabetic retina and the direct monitoring of its
functional and anatomic consequences. These studies are un-
derway.
References
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1994;145:574–584.
2. AdamisAP,ShimaDT,TolentinoMJ,etal.Inhibitionofvascular
endothelialgrowthfactorpreventsretinalischemia-associatediris
neovascularization in a nonhuman primate. Arch Ophthalmol.
FIGURE4. RatretinaICAM-1RNaseprotectionassays2.5hoursafter 1996;114:66–71.
intravitreal injection of VEGF or TNF-(cid:97). The contralateral eyes were 3. Aiello LP, Pierce EA, Foley ED, et al. Suppression of retinal neo-
injected with PBS. These are representative data from 1 of 4 rats vascularizationinvivobyinhibitionofvascularendothelialgrowth
injectedwithVEGFand1of5ratsinjectedwithTNF-(cid:97).Graphshows factor(VEGF)usingsolubleVEGF-receptorchimericproteins.Proc
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