Cell cycle and apoptosis genes in atherosclerosis Boesten, Lianne Simone Mirjam
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(2) &KDSWHU. 0DFURSKDJHSDQG DWKHURVFOHURVLV.
(3)
(4) -ACROPHAGE P CONTROLS FOAM CELL DEATH IN ATHEROSCLEROTIC LESIONS OF APOLIPOPROTEIN % DElCIENT MICE ,IANNE 3- "OESTEN
(5) ! 3USANNE - :ADELAAR
(6) !NITA VAN .IEUWKOOP
(7) ,IHUI (U !MINA &!3 4EUNISSE !ART ' *OCHEMSEN "ASTIAAN %VERS -ARION ** 'IJBELS
(8) "ART *- VAN 6LIJMEN ,OUIS - (AVEKES
(9)
(10) -ENNO 0* DE 7INTHER $EPT OF 'ENERAL )NTERNAL -EDICINE
(11) $EPT OF #ARDIOLOGY
(12) $EPT OF -OLECULAR AND #ELL "IOLOGY
(13) (EMOSTASIS AND 4HROMBOSIS 2ESEARCH #ENTER
(14) $EPT OF (EMATOLOGY
(15) ,EIDEN 5NIVERSITY -EDICAL #ENTER
(16) ,EIDEN
(17) 4HE .ETHERLANDS
(18) 4./ 1UALITY OF ,IFE 'AUBIUS ,ABORATORY
(19) ,EIDEN 4HE .ETHERLANDS
(20) 4HE .ETHERLANDS #ANCER )NSTITUTE
(21) !MSTERDAM
(22) 4HE .ETHERLANDS
(23) $EPARTMENT OF -OLECULAR 'ENETICS
(24) #ARDIOVASCULAR 2ESEARCH )NSTITUTE -AASTRICHT
(25) $EPARTMENT OF 0ATHOLOGY
(26) -AASTRICHT 5NIVERSITY
(27) -AASTRICHT
(28) 4HE .ETHERLANDS. $%675$&7 4HE CELLULAR COMPOSITION OF AN ATHEROSCLEROTIC LESION IS DETERMINED BY MANY FAC TORS INCLUDING CELL INlLTRATION
(29) PROLIFERATION AND CELL DEATH
(30) EITHER BY APOPTOSIS OR NECROSIS 4HE TUMOR SUPPRESSOR GENE P HAS BEEN SHOWN TO REGULATE BOTH CELL PROLIFERATION AND CELL DEATH IN MANY CELL TYPES 4O STUDY THE ROLE OF MACROPHAGE P IN THE DEVELOPMENT OF ATHEROSCLEROSIS
(31) WE GENERATED APO% DElCIENT MICE WITH A MACROPHAGE RESTRICTED DELETION OF P ,YS-#RE PLOX0LOX0 APO% MICE AND CON TROL LITTERMATES PLOX0LOX0 APO% MICE AND ANALYZED EARLY AND ADVANCED ATHEROSCLE ROSIS DEVELOPMENT !BSENCE OF MACROPHAGE P DID NOT AFFECT LESION AREA IN BOTH EARLY AND ADVANCED ATHEROSCLEROSIS
(32) NEITHER IN THE AORTIC ROOT NOR IN THE AORTIC ARCH AND THORACIC AORTA )N EARLY ATHEROSCLEROSIS
(33) ABSENCE OF MACROPHAGE P
(34) RESULTED IN REDUCED APOPTOSIS
(35) HOWEVER WITHOUT CHANGES IN LESION COMPOSITION )N CONTRAST
(36) IN ADVANCED ATHEROSCLEROSIS
(37) REDUCED APOPTOSIS UPON ABSENCE OF MACROPHAGE P
(38) COINCIDED WITH INCREASED NECROTIC DEATH
(39) INCREASED FOAM CELL CONTENT
(40) AND REDUCED LIPID CORE FORMATION 0ROLIFERATION WAS NOT AFFECTED BY THE ABSENCE OF MACROPHAGE P IN BOTH EARLY AND ADVANCED ATHEROSCLE ROSIS (ENCE
(41) OUR DATA POINT TOWARDS AN IMPORTANT ROLE FOR MACROPHAGE P IN INDUC TION OF FOAM CELL APOPTOSIS AND PREVENTION OF LESIONAL NECROSIS .
(42) #HAPTER . !. . THEROSCLEROSIS IS AN INmAMMATORY DISEASE OF THE LARGE VESSELS IN WHICH MAC ROPHAGES PLAY A CENTRAL ROLE
(43) !CCUMULATION OF MACROPHAGE FOAM CELLS IN THE VESSEL WALL RESULTS IN THE FORMATION OF FATTY STREAKS 4HESE LESIONS MAY BE REVERSIBLE AND MAY NOT CAUSE CLINICAL SYMPTOMS (OWEVER
(44) MACROPHAGE ACCU MULATION WITHIN THE ARTERIAL INTIMA SETS THE STAGE FOR PROGRESSION OF THE ATHEROMA AND EVOLUTION INTO MORE COMPLICATED LESIONS THAT CAN CAUSE CLINICAL SYMPTOMS BY EVENTUAL RUPTURE OR EROSION OF THE PLAQUE #HANGES IN THE CELLULAR COMPOSITION OF AN ATHEROSCLEROTIC LESION ARE IMPORTANT IN MODULATING THE RISK OF ACUTE CORONARY SYNDROMES #ELL PROLIFERATION AND CELL DEATH ARE CRUCIAL PROCESSES IN REGULATING CELL NUMBERS IN THE ATHEROSCLEROTIC LESION AND MAY THEREBY DIRECTLY INmUENCE LESION COMPOSITION AND STABILITY 4HE P TUMOR SUPPRESSOR PROTEIN IS AN ESSENTIAL GENE IN CELL PROLIFERATION AND CELL DEATH AND PLAYS A PIVOTAL ROLE IN THE CELLULAR RESPONSE TO A RANGE OF ENVIRONMENTAL AND INTRACELLULAR STRESS SIGNALS -UTATIONS IN P OCCUR IN ABOUT HALF OF THE HUMAN CANCERS
(45) RESULTING IN LOSS OF APOPTOTIC FUNCTION 0 IS A POTENT TRANSCRIPTION FACTOR
(46) PREDOMINANTLY ACTING IN THE ' PHASE OF CELL CYCLE PROGRESSION
(47) REGULATING MULTIPLE DOWNSTREAM GENES IMPLICATED IN CELL CYCLE CONTROL
(48) APOPTOSIS
(49) DIFFERENTIATION
(50) AND SENESCENCE
(51) )N ATHEROSCLEROSIS
(52) P WAS IMMUNOHISTOCHEMICALLY VISUALIZED IN HU MAN CAROTID ATHEROMATOUS LESIONS IN VIRTUALLY ALL CELL TYPES MACROPHAGES
(53) SMOOTH MUSCLE CELLS
(54) ENDOTHELIAL CELLS 2ECENT MOUSE STUDIES DEMONSTRATED THAT P PLAYS AN IMPORTANT ROLE IN THE PROGRESSION OF ATHEROSCLEROTIC LESIONS 7HOLE BODY P INACTIVATION IN APOLIPOPROTEIN % DElCIENT APO% MICE ACCELERATED ATHEROSCLEROSIS PRIMARILY BY INCREASED CELLULAR PROLIFERATION )N ADDITION
(55) USING BONE MARROW TRANS PLANTATION IN BOTH APO% ,EIDEN AND ,$, RECEPTOR DElCIENT ,$,2 MICE IT WAS SHOWN THAT P OF HEMATOPOIETIC ORIGIN IS
(56) AT LEAST IN PART
(57) RESPONSIBLE FOR THE INHIBI TION OF ATHEROGENESIS IN THESE MODELS (ENCE
(58) THESE STUDIES SHOW AN IMPORTANT ROLE FOR BOTH MACROPHAGE AND LYMPHOCYTE DERIVED P OR IN ADDITION THEIR INTERPLAY IN THE DEVELOPMENT OF ATHEROSCLEROSIS 4O DISTINGUISH THE EFFECTS OF P DElCIENCY SPECIlCALLY IN MACROPHAGES FROM PROCESSES AFFECTED BY LYMPHOCYTE P DElCIENCY
(59) WE EMPLOYED A CONDITIONAL DELE TION APPROACH USING THE #RE LOX0 SYSTEM -ACROPHAGE SPECIlC P DELETION WAS AC COMPLISHED BY COMBINING MICE CARRYING A P ALLELE THAT WAS mANKED BY LOX0 SITES WITH ,YS-#RE MICE %XPRESSION OF #RE IN THE MYELOID LINEAGE IN THE ,YS-#RE MICE WILL RESULT IN CELL SPECIlC DELETION OF P AND THEREBY GIVE MACROPHAGES THAT LACK P 5SING THIS APPROACH
(60) WE FOUND THAT ABSENCE OF MACROPHAGE P DID NOT AFFECT LESION AREA IN BOTH EARLY AND ADVANCED ATHEROSCLEROSIS
(61) NEITHER IN THE AORTIC ROOT NOR IN THE AORTIC ARCH AND THORACIC AORTA )N EARLY ATHEROSCLEROSIS
(62) ABSENCE OF MACRO PHAGE P RESULTED IN REDUCED APOPTOSIS
(63) HOWEVER WITHOUT CHANGES IN LESION COM POSITION )N CONTRAST
(64) IN ADVANCED ATHEROSCLEROSIS
(65) REDUCED APOPTOSIS DUE TO ABSENCE OF MACROPHAGE P
(66) COINCIDED WITH INCREASED NECROTIC DEATH
(67) INCREASED FOAM CELL CONTENT AND REDUCED LIPID CORE FORMATION 4HESE STUDIES INDICATE THAT MACROPHAGE P IS PRIMARILY INVOLVED IN DETERMINING ATHEROSCLEROTIC LESION COMPOSITION BY CON TROLLING THE BALANCE OF LESIONAL APOPTOSIS AND NECROSIS .
(68) . -ACROPHAGE P AND ATHEROSCLEROSIS . 0(7+2'6 0LFHDQGGLHW 4HE EXPERIMENTAL ANIMALS WERE OBTAINED BY COMBINING MICE CARRYING THE mOXED P GENE WITH ,YS-#RE MICE A GENEROUS GIFT FROM $R "% #LAUSEN
(69) !-#
(70) 4HE .ETHERLANDS AND $R ) &ORSTER
(71) 5NIVERSITY OF #OLOGNE
(72) 'ERMANY
(73) AND APO% DElCIENT MICE RESULTING IN MICE THAT ARE HOMOZYGOUSLY mOXED FOR P AND DElCIENT FOR APO% AND THAT EITHER EXPRESS #RE IN THEIR MACROPHAGES ,YS-#RE PLOX0LOX0 APO % FURTHER REFERRED TO AS PDEL OR DO NOT EXPRESS #RE AND REMAIN WILDTYPE FOR P PLOX0LOX0 APO% FURTHER REFERRED TO AS Pm -ICE WERE GENOTYPED BY POLYMERASE CHAIN REACTION 0#2 FOR ,YS-#RE
(74) PLOX0LOX0
(75) AND APO% STATUS &OR EXPERIMENTS
(76) WEEK OLD MALE PDEL AND LITTERMATE CONTROL Pm MICE WERE USED -ICE WERE FED A SEMI SYNTHETIC CHOLESTEROL RICH DIET COMPOSED ESSENTIALLY ACCORDING TO .ISHINA ET AL SUPPLEMENTED WITH COCOA BUTTER
(77) BY WEIGHT AND CHOLESTEROL
(78) BY WEIGHT
(79) WITHOUT CHOLATE (OPE &ARMS
(80) 7OERDEN
(81) 4HE .ETHERLANDS 4HE ANIMALS WERE FED THE CHOLESTEROL RICH DIET FOR EITHER WEEKS EARLY ATHEROSCLEROSIS DEVELOP MENT
(82) N AND N FOR PDEL AND Pm MICE
(83) RESPECTIVELY OR WEEKS ADVANCED ATHEROSCLEROSIS DEVELOPMENT
(84) N AND N FOR PDEL AND Pm MICE
(85) RESPECTIVELY -ICE WERE GIVEN FOOD AND WATER AD LIBITUM !LL ANIMAL WORK WAS APPROVED BY INSTI TUTIONAL REGULATORY AUTHORITY AND CARRIED OUT IN COMPLIANCE WITH GUIDELINES ISSUED BY THE $UTCH GOVERNMENT 4XDQWL¿FDWLRQRIPDFURSKDJHSE\:HVWHUQEORWWLQJ 0ERITONEAL MACROPHAGES WERE OBTAINED FROM PDEL AND Pm MICE FOUR DAYS AFTER INTRAPERITONEAL INJECTION OF ML THIOGLYCOLLATE BROTH WTVOL BY mUSHING THE PERITONEUM WITH ML ICE COLD 0"3 -ACROPHAGES WERE WASHED WITH 20-) CONTAINING FOETAL CALF SERUM AND THE CELLS OF EACH MOUSE WERE SUBSEQUENTLY DIVIDED OVER TWO CM CULTURE PLATES !FTER HOURS THE DUPLICATE DISHES WERE EITHER MOCK TREATED OR INCUBATED WITH A COMBINATION OF ETOPOSIDE -
(86) 3IGMA !LDRICH AND PROTEASOME INHIBITOR -' -
(87) 3IGMA !LDRICH FOR HOURS 3UBSEQUENTLY
(88) NON ADHERENT CELLS WERE REMOVED BY WASHING TWICE WITH ICE COLD 0"3
(89) AND CELLS WERE HARVESTED IN 'IORDANO BUFFER M- 4RIS (#L
(90) P( M- .A#L 4RITON 8 M- %$4!
(91) SUPPLEMENTED WITH PROTEASE INHIBITORS AND ANALYZED BY 7ESTERN BLOT AS DESCRIBED PREVIOUSLY ,YSATES FROM MOUSE EMBRYO lBROBLAST DERIVED FROM WILD TYPE MICE OR HOMOZYGOUS PMDM DElCIENT DOUBLE KNOCK OUT $+/ MICE WERE USED AS POSITIVE AND NEGATIVE CONTROLS
(92) RESPECTIVELY "LOTS WERE INCUBATED WITH ANTI P !B -ERCK "IOCHEMICALS AND AFTER STRIPPING WITH ANTI D4UBULIN #LONE$-! 3IGMA !LDRICH AS LOADING CONTROL 0ROTEIN BANDS WERE DE TECTED BY ENHANCED CHEMILUMINESCENCE WITH THE USE OF THE 3UPER3IGNAL 7EST $URA KIT 0IERCE
(93) VISUALIZED BY AUTORADIOGRAPHY OR BY IMAGING WITH THE #HEMI'ENIUS 8% 3YNGENE
(94) #AMBRIDGE
(95) 5+ 1UANTIlCATION OF THE P AND TUBULIN PROTEIN LEVELS WAS PERFORMED WITH USE OF THE 3YNGENE 'ENE4OOLS SOFTWARE %ORRGVDPSOLQJDQGDQDO\VLV "LOOD SAMPLES WERE COLLECTED
(96) AFTER HOURS FASTING
(97) IN %$4! COATED VIALS 3ARSTEDT
(98) .
(99) #HAPTER . .àMBRECHT
(100) 'ERMANY BY BLEEDING FROM THE TAIL VEIN 0LASMA CHOLESTEROL AND TRIGLYC ERIDE LEVELS WERE MEASURED ENZYMATICALLY USING COMMERCIALLY AVAILABLE KITS 2OCHE $IAGNOSTICS 'MB(
(101) -ANNHEIM
(102) 'ERMANY 4OTAL BLOOD LEUKOCYTE #$
(103) 4 CELL #$
(104) " CELL #$ AND MYELOID #$B NUMBERS WERE DETERMINED BY &!#3 ANALYSIS &!#3#ALIBUR
(105) "$ "IOSCIENCES
(106) #ALIFORNIA
(107) 53! FOLLOWING STANDARD PROTOCOL 4RU#/5.4
(108) "$ "IOSCIENCES
(109) #ALIFORNIA
(110) 53!
(111) AS DESCRIBED BEFORE . . $WKHURVFOHURVLVDQDO\VLV !FTER EITHER OR WEEKS ON A CHOLESTEROL RICH DIET MICE WERE SACRIlCED (EARTS WERE OVERNIGHT lXED IN FORMALIN P( AND EMBEDDED IN PARAFlN 4HE AORTA WAS SNAP FROZEN AND STORED AT # 4O QUANTIFY CROSS SECTIONAL LESION AREA IN THE AORTIC ROOT FORMALIN lXED HEARTS WERE PROCESSED AS DESCRIBED BEFORE
(112) 3ECTIONS OF THE AORTIC ROOT WERE ROUTINELY STAINED WITH HEMATOXYLIN PHLOXINE SAFFRAN (03 FOR MOR PHOMETRIC ANALYSIS !REAS WERE DETERMINED USING ,EICA 1WIN IMAGE SOFTWARE %)3
(113) !SBURY
(114) .* &OR EN FACE ANALYSIS OF LESION AREA IN THE AORTIC ARCH AND THORACIC AORTA
(115) THE AORTA WAS CLEANED IN SITU FROM PERIADVENTITIAL TISSUE
(116) DISSECTED FROM THE AORTIC ARCH DOWN TO THE ILIAC BIFURCATION
(117) OPENED LONGITUDINALLY
(118) PINNED ON A SILICONE BASEMENT AND STAINED WITH /IL 2ED / 4HE PERCENTAGE OF SURFACE AREA COVERED BY ATHEROSCLEROTIC LESIONS /IL 2ED / POSITIVE AREA WAS QUANTIlED STARTING FROM THE TOP OF THE AORTIC ARCH CM DOWN TOWARDS THE THORACIC AORTA BY COMPUTER ASSISTED ANALYSIS N VS N FOR EARLY ATHEROSCLEROSIS AND N VS N FOR ADVANCED ATHEROSCLEROSIS FOR PDEL AND Pm MICE
(119) RESPECTIVELY !LL ANALYSES WERE PERFORMED DOUBLE BLINDLY WITH OUT PRIOR KNOWLEDGE OF THE GENOTYPE /HVLRQFRPSRVLWLRQDQDO\VLV &OR COMPOSITIONAL ANALYSIS OF THE LESIONS LIPID CORE
(120) NECROSIS AND MACROPHAGE CON TENT WERE DETERMINED ,IPID CORE AREA WAS DElNED BY THE PRESENCE OF CHOLESTEROL CLEFTS
(121) EXTRACELLULAR LIPIDS AND THE COMPLETE ABSENCE OF NUCLEI )N ADDITION
(122) NECROSIS WAS DElNED BY THE PRESENCE OF PYKNOSIS
(123) KARYORRHEXIS
(124) OR COMPLETE ABSENCE OF NU CLEI ,IPID CORE AREA AND NECROSIS AREA WERE MEASURED USING MORPHOMETRIC ANALY SIS
(125) AS DESCRIBED ABOVE 3ERIAL SECTIONS WERE STAINED FOR MACROPHAGES USING A RABBIT ANTIBODY TO MOUSE MACROPHAGES !)!
(126)
(127) !CCURATE #HEMICAL AND 3CI ENTIlC !)! POSITIVE AREAS WERE QUANTIlED AS DESCRIBED BEFORE 4O LABEL $.! SYNTHESIZING CELLS THE MICE RECEIVED "ROMO $EOXYURIDINE "RD5
(128) 3IGMA MGKG
(129) INTRAPERITONEALLY FOR CONSECUTIVE DAYS PRIOR TO SACRIlCA TION 3ECTIONS WERE STAINED FOR PROLIFERATION USING A MONOCLONAL MOUSE ANTI "RD5 ANTIBODY $!+/ !3 $ENMARK AND FOR APOPTOSIS USING THE 45.%, TECHNIQUE AC CORDING TO MANUFACTURERS PROTOCOL )N SITU CELL DETECTION KIT 0/$
(130) 2OCHE $IAGNOS TICS 'MB(
(131) -ANNHEIM
(132) 'ERMANY .UMBERS OF "RD5 OR 45.%, POSITIVE NUCLEI WERE EXPRESSED PER TOTAL ATHEROSCLEROTIC LESION AREA
(133) CORRECTED FOR LIPID CORE AREA
(134) AS THE LIPID CORE IS AN ACELLULAR AREA OF THE ATHEROSCLEROTIC LESION WITHOUT NUCLEI AND THERE FORE BY DElNITION DOES NOT CONTAIN ANY "RD5 OR 45.%, POSITIVE NUCLEI 6WDWLVWLFDODQDO\VLV 3TATISTICAL ANALYSES WERE PERFORMED USING 'RAPHPAD 0RISM !LL DATA GROUPS WERE .
(135) . -ACROPHAGE P AND ATHEROSCLEROSIS . lRST TESTED FOR NORMALITY )F THE DATA WERE DISTRIBUTED NORMALLY
(136) GROUPS WERE COM PARED USING 7ELCHS CORRECTED T TEST )F DATA WERE NOT DISTRIBUTED NORMALLY
(137) GROUPS WERE COMPARED USING -ANN 7HITNEY RANK SUM TEST $ATA ARE EXPRESSED AS MEAN3$ 0 VALUE WAS REGARDED AS SIGNIlCANT 5(68/76 *HQHUDOFKDUDFWHULVWLFVRIDSR(GH¿FLHQWSGHOPLFH ,ITTERMATE MALE APO% MICE EITHER LACKING P IN THEIR MACROPHAGES PDEL OR WILDTYPE FOR P Pm WERE FED A CHOLESTEROL RICH DIET FOR WEEKS EARLY ATHERO SCLEROSIS OR WEEKS ADVANCED ATHEROSCLEROSIS $URING THE STUDY
(138) THE MICE AP PEARED HEALTHY AND DISPLAYED NO SIGNS OF ABNORMALITIES !S SHOWN IN 4ABLE
(139) BOTH AFTER AND WEEKS OF A CHOLESTEROL RICH DIET CHALLENGE
(140) MEAN BODY WEIGHT WAS NOT DIFFERENT BETWEEN PDEL AND Pm MICE 0LASMA CHOLESTEROL
(141) TRIGLYCERIDE AND HEMATOCRITE LEVELS 4ABLE AND LIPOPROTEIN PROlLES DATA NOT SHOWN DID NOT DIFFER BETWEEN PDEL AND Pm MICE -OREOVER
(142) ABSENCE OF MACROPHAGE P DID NOT AFFECT CIRCULATING 4 CELL
(143) " CELL OR MYELOID CELL CONCENTRATIONS 4ABLE AS ANALYZED AFTER WEEKS FEEDING A CHOLESTEROL RICH DIET 4ABLE 'ENERAL CHARACTERISTICS OF MALE PDEL AND Pm MICE AFTER FEEDING A CHOLESTEROL RICH DIET FOR WEEKS EARLY ATHEROSCLEROSIS DEVELOPMENT OR WEEKS ADVANCED ATHEROSCLEROSIS DEVELOPMENT Pm !THEROSCLEROSIS DEVELOPMENT 7EIGHT G 0LASMA LIPID LEVELS MMOL, (EMATOCRITE "LOOD LEUKOCYTES CELLSM,. PDEL %ARLY. Pm. PDEL. !DVANCED. #HOLESTEROL. . . . . 4RIGLYCERIDES. . . . . . . . . ND. ND. . . ND. ND. . . ND. ND. . . 4 CELLS #$ . " CELLS #$ . -YELOID CELLS #$B ND NOT DETERMINED. 4O CONlRM DELETION OF P IN MACROPHAGES
(144) WE ANALYZED P PROTEIN LEVELS IN THIO GLYCOLLATE ELICITED MACROPHAGES USING 7ESTERN BLOT ANALYSIS 9IELDS OF THIOGLYCOLLATE ELICITED PERITONEAL MACROPHAGES FROM PDEL AND Pm MICE WERE SIMILAR DATA NOT SHOWN 7ESTERN BLOT ANALYSIS SHOWED THAT P WAS VIRTUALLY ABSENT IN THIOGLYCOL LATE ELICITED MACROPHAGES FROM PDEL ANIMALS
(145) WHILE IT COULD EASILY BE DETECTED IN Pm MACROPHAGES &IGURE ! 4O FURTHER INCREASE P SIGNALS IN THE MACROPHAGES
(146) WE TREATED THE CELLS WITH THE $.! DAMAGING AGENT ETOPOSIDE IN THE PRESENCE OF THE PROTEASOME INHIBITOR -' 3UCH TREATMENTS ARE KNOWN TO INCREASE THE LEV ELS OF THE VERY UNSTABLE P PROTEIN !S CAN BE SEEN IN lGURE !
(147) THIS TREATMENT STRONGLY INCREASED P LEVELS IN Pm MACROPHAGES AND RESULTED IN THE PRESENCE OF .
(148) . %&*. . . . '* . . . . . . 1-.-0)%&
(149) . 2#2*),. . . . %&*. . '*. .. 2#2*),. #HAPTER . . . . . &*"1)3&. *&3&*. &IGURE ! 0ROTEIN EXTRACTS FROM PDEL AND Pm MACROPHAGES
(150) EITHER MOCK TREATED OR TREATED WITH ETOPOSIDE AND PROTEASOME INHIBITOR -'
(151) WERE ANALYZED FOR P AND D TUBULIN EXPRESSION " P PROTEIN EXPRESSION LEVELS IN LYSATES FROM REPRESENTATIVE PDEL AND Pm MACROPHAGES TREATED WITH ETOPOSIDE AND -' 0 PROTEIN LEVELS IN PDEL MACROPHAGES WERE CORRELATED TO THE P EXPRESSION LEVELS IN Pm MACROPHAGES
(152) CORRECTED FOR THE LOADING CONTROL D TUBULIN -%& LYSATE FROM WILD TYPE MOUSE EMBRYO lBROBLASTS POSITIVE CONTROL $+/ LYSATE FROM HOMOZYGOUS PMDM DOUBLE KNOCK OUT MOUSE EMBRYO lBROBLASTS NEGATIVE CONTROL. A P BAND IN THE PDEL CELLS
(153) INDICATING THAT SOME REMAINING P WAS LEFT 3EMI QUANTITATIVE 0#2 CONlRMED THAT SOME WILD TYPE ALLELE WAS LEFT IN THE PDEL MACRO PHAGES DATA NOT SHOWN 4HE RELATIVE DIFFERENCE IN P LEVELS BETWEEN PDEL AND Pm MACROPHAGES WAS QUANTIlED USING 7ESTERN BLOTS OF ETOPOSIDE-' TREATED CELLS AND SHOWED A REDUCTION OF APPROXIMATELY IN P PROTEIN LEVELS IN PDEL MACROPHAGES AS COMPARED TO Pm MICE &IGURE " -OREOVER
(154) TITRATION ON 7ESTERN BLOT USING LYSATES FROM Pm MACROPHAGES TO OBTAIN P PROTEIN LEVELS SIMILAR TO THAT OF PDEL MACROPHAGES ALSO REQUIRED OVER FOLD DILUTION CONlRMING THE REDUCTION IN P PROTEIN LEVELS DATA NOT SHOWN 4HESE DATA SHOW THAT CELL SPECIlC DELETION OF P RESULTS IN A STRONG REDUCTION IN P PROTEIN LEVELS IN MAC ROPHAGES $QDO\VLVRIDWKHURVFOHURWLFOHVLRQDUHD -ICE FED THE CHOLESTEROL RICH DIET FOR WEEKS EARLY ATHEROSCLEROSIS OR WEEKS ADVANCED ATHEROSCLEROSIS WERE SACRIlCED FOR COLLECTION OF HEART
(155) AORTA
(156) AND OTHER ORGANS -ORPHOMETRIC ANALYSIS OF THE TOTAL ATHEROSCLEROTIC LESION AREA IN THE AORTIC ROOT SHOWED NO DIFFERENCE BETWEEN PDEL AND Pm IN EARLY LESION SIZE 0
(157) &IGURE ! AND " AND IN ADVANCED LESION SIZE 0
(158) &IGURE ! AND " )N ADDI TION
(159) EN FACE ANALYSIS OF /IL 2ED / STAINED AORTAS DID ALSO NOT REVEAL A DIFFERENCE IN RELATIVE LESION AREA BETWEEN PDEL AND Pm MICE ON EARLY 0
(160) &IGURE ! AND " AND ADVANCED ATHEROSCLEROSIS DEVELOPMENT 0
(161) &IGURE ! AND " (ENCE
(162) .
(163) . %3",$&%"1(&/-0$*&/-0)0. "/*6"1(&/-0$*&/-0)0
(164) . &0)-,"/&" 5 ȝ+
(165) . &0)-,"/&" 5 ȝ+
(166) . . -ACROPHAGE P AND ATHEROSCLEROSIS .
(167). . . . . '* . '*. . %&*. . . .
(168). . '*. . %&*. . %&*. . '*. . %&*. . . . &IGURE !ORTIC ROOT ATHEROSCLEROSIS IN PDEL AND Pm MICE ! %ARLY AND ADVANCED AORTIC ROOT ATHEROSCLEROSIS IN Pm CLOSED CIRCLES AND PDEL OPEN CIRCLES MICE 3YMBOLS INDICATE INDIVIDUAL MICE ,INE REPRESENTS MEAN AREA FOR EACH GROUP " 2EPRESENTATIVE PHOTOMICROGRAPHS OF ATHEROSCLEROTIC LESIONS IN Pm AND PDEL MICE 3ERIAL SECTIONS WERE STAINED WITH (03 , LIPID CORE
(169) MAGNIlCATION X
(170) SCALE BAR M . "/*6"1(&/-0$*&/-0)0 /&"$-3&/&%4)1( *&0)-,0. /&"$-3&/&%4)1( *&0)-,0. . .
(171) . . %3",$&%"1(&/-0$*&/-0)0. . '*. . %&*. . '*. . %&*. .
(172). . '*. . %&*. . '*. . %&*. &IGURE !ORTIC ARCH AND THORACIC AORTA ATHEROSCLEROSIS IN PDEL AND Pm MICE ! %ARLY AND ADVANCED ATHEROSCLEROSIS IN Pm CLOSED TRIANGLES AND PDEL OPEN TRIANGLES MICE 3YMBOLS INDICATE INDIVIDUAL MICE ,INE REPRESENTS MEAN AREA FOR EACH GROUP " 2EPRESENTATIVE /IL 2ED / STAINED AORTAS OF Pm AND PDEL MICE IN EARLY AND ADVANCED ATHEROSCLEROSIS DEVELOPMENT .
(173) ABSENCE OF MACROPHAGE P DID NOT AFFECT TOTAL ATHEROSCLEROTIC LESION AREA
(174) BOTH IN EARLY AND ADVANCED ATHEROSCLEROTIC LESIONS AT TWO DIFFERENT REGIONS IN THE AORTAS OF APO% DElCIENT MICE &HOOSUROLIHUDWLRQDQGFHOOGHDWK 4O INVESTIGATE WHETHER MACROPHAGE P DElCIENCY AFFECTS CELL TURNOVER IN THE LE SIONS BOTH CELL PROLIFERATION AND CELL DEATH WERE FOLLOWED DURING ATHEROSCLEROSIS DEVELOPMENT &OR ANALYSIS OF CELL PROLIFERATION MICE WERE INJECTED DAILY WITH "RD5 FOR DAYS BEFORE THE END OF THE EXPERIMENT )N BOTH THE EARLY AND ADVANCED ATHERO SCLEROSIS GROUP THE INCIDENCE OF "RD5 CELLS DID NOT DIFFER BETWEEN PDEL AND Pm MICE &IGURE !
(175) INDICATING THAT MACROPHAGE P DElCIENCY DID NOT AFFECT LESIONAL PROLIFERATION IN BOTH EARLY AND ADVANCED ATHEROSCLEROSIS "/*6 "1(&/-0$*&/-0)0. /%! $&**0 ȝ+
(176). .
(177) . . '*. . %&*. .
(178) . . .
(179) . . . . .
(180) . . '*. . '*. . %&*. . %&*. . . . .
(181) . . . %&*. . . . &$/-0)0 -'1-1"**&0)-,"/&". . . '*. . .
(182) . . %3",$&% "1(&/-0$*&/-0)0. ! $&**0 ȝ+
(183). /%! $&**0 ȝ+
(184). . ! $&**0 ȝ+
(185). . . . &$/-0)0 -'1-1"**&0)-,"/&". #HAPTER . . . '*. . . %&*. . . .
(186) . . . '*. . %&*. &IGURE 0ROLIFERATION
(187) APOPTOSIS AND NECROSIS IN Pm AND PDEL MICE ! 0ROLIFERATION WAS DETECTED BY "RD5 STAINING "ARS REPRESENT THE NUMBER OF "RD5 NUCLEI PER LESION AREA " !POPTOSIS WAS DETECTED BY 45.%, STAINING "ARS REPRESENT THE NUMBER OF 45.%, NUCLEI PER LESION AREA # .ECROSIS
(188) DElNED BY THE PRESENCE OF PYKNOSIS
(189) KARYORRHEXIS
(190) OR COMPLETE ABSENCE OF NUCLEI
(191) WAS ANALYZED ON (03 SECTIONS "ARS REPRESENT NECROTIC AREA PER LESION AREA "LACK BARS
(192) Pm MICE WHITE BARS PDEL MICE %RROR BARS INDICATE 3%- 0 .
(193) . -ACROPHAGE P AND ATHEROSCLEROSIS . 4O INVESTIGATE CELL DEATH IN THE ATHEROSCLEROTIC LESIONS APOPTOSIS AND NECROSIS WERE QUANTIlED !POPTOTIC CELLS IN THE ATHEROSCLEROTIC LESIONS WERE IDENTIlED AS 45.%, POSITIVE CELLS 3TRIKINGLY
(194) THE INCIDENCE OF 45.%, POSITIVE NUCLEI WAS STRONGLY REDUCED IN PDEL MICE IN BOTH EARLY AND ADVANCED ATHEROSCLEROSIS DEVELOPMENT AND
(195) RESPECTIVELY
(196) 0
(197) &IGURE " )N ADDITION TO APOPTOSIS
(198) NECROSIS IN THE LESIONS WAS QUANTIlED .ECROSIS IN THE ADVANCED ATHEROSCLEROSIS GROUP WAS FOLD INCREASED IN THE PDEL GROUP
(199) AS COMPARED TO THE CONTROLS 0
(200) &IGURE # ! SIMILAR TREND WAS OBSERVED IN EARLY ATHEROSCLEROSIS ALTHOUGH NOT SIGNIlCANT &IGURE # 4HESE ANALYSES SHOW THAT MACROPHAGE P DELETION PREVENTS LESIONAL MACRO PHAGE APOPTOSIS BOTH IN EARLY AND ADVANCED ATHEROSCLEROSIS )N ADDITION
(201) PREVENTION OF MACROPHAGE APOPTOSIS IN ADVANCED ATHEROSCLEROSIS SWITCHES THE CELL DEATH PATH WAY TOWARDS NECROTIC CELL DEATH 0DFURSKDJHVDQGOLSLGFRUH 4O ANALYZE WHETHER THE CHANGES IN LESIONAL CELL DEATH IN PDEL MICE COINCIDED WITH CHANGES IN ATHEROSCLEROTIC LESION COMPOSITION WE PERFORMED A MORE DETAILED PHE NOTYPIC ANALYSIS OF THE LESIONS -ACROPHAGE AREA WAS DETECTED BY IMMUNOSTAINING AND LIPID CORE AREA WAS DElNED BY THE PRESENCE OF CHOLESTEROL CLEFTS
(202) EXTRACELLULAR LIPIDS AND COMPLETE ABSENCE OF NUCLEI )N THE EARLY ATHEROSCLEROSIS GROUP BOTH MAC ROPHAGE AREA AND LIPID CORE CONTENT WERE NOT AFFECTED BY MACROPHAGE RESTRICTED P DELETION 4ABLE (OWEVER
(203) QUANTIlCATION OF MACROPHAGE AREA IN THE ADVANCED ATHEROSCLEROSIS GROUP REVEALED A INCREASE IN PDEL MICE AS COMPARED TO THE Pm CONTROL MICE 0
(204) 4ABLE )NTERESTINGLY
(205) THESE CHANGES COINCIDED WITH A STRONG REDUCTION IN THE LIPID CORE CONTENT OF THE LESIONS IN PDEL MICE AS COM PARED TO Pm CONTROL MICE 0
(206) 4ABLE
(207) &IGURE "
(208) RIGHT PANEL (ENCE
(209) THE DE CREASE IN LESIONAL APOPTOSIS IN PDEL MICE COINCIDES WITH A CHANGE IN CELLULAR LESION COMPOSITION TOWARDS ATHEROSCLEROTIC LESIONS WITH AN INCREASED MACROPHAGE AREA AND A DECREASED LIPID CORE 4ABLE #HARACTERISTICS OF EARLY WEEKS CHOLESTEROL RICH DIET AND ADVANCED WEEKS CHOLESTEROL RICH DIET ATHEROSCLEROTIC LESIONS IN PDEL AND Pm MICE Pm !THEROSCLEROSIS DEVELOPMENT ,ESION MACROPHAGE AREA OF TOTAL LESION AREA ,IPID CORE OF TOTAL LESION AREA. PDEL %ARLY. Pm. PDEL. !DVANCED. . . . . . . . . 0 . ',6&866,21 )N THE PRESENT STUDY
(210) WE INVESTIGATED THE ROLE OF MACROPHAGE P IN THE PATHOGEN ESIS OF ATHEROSCLEROSIS !BSENCE OF MACROPHAGE P DID NOT AFFECT LESION AREA OF EARLY AND ADVANCED ATHEROSCLEROSIS ANALYZED BOTH AT THE LEVEL OF THE AORTIC ROOT AND AT THE LEVEL OF THE AORTIC ARCH AND THORACIC AORTA IN !PO% DElCIENT MICE (OWEVER
(211) DETAILED ANALYSIS OF ATHEROSCLEROSIS IN THE AORTIC ROOT REVEALED THAT APOPTOSIS OF FOAM .
(212) #HAPTER . CELLS WAS STRONGLY REDUCED IN PDEL MICE AT BOTH TIME POINTS
(213) WHILE PROLIFERATION WAS UNAFFECTED -OREOVER
(214) THE REDUCTION IN LESIONAL APOPTOSIS IN PDEL MICE COINCIDED WITH INCREASED NECROSIS IN THE ADVANCED ATHEROSCLEROTIC LESIONS 4HE CHANGES IN LE SIONAL CELL DEATH WERE ACCOMPANIED BY AN INCREASE IN RELATIVE MACROPHAGE AREA AND A DECREASE IN RELATIVE LIPID CORE AREA IN THE ADVANCED ATHEROSCLEROSIS GROUP (ENCE
(215) THESE STUDIES DEMONSTRATE THAT MACROPHAGE P IS A MAJOR MEDIATOR OF FOAM CELL APOPTOSIS AND INHIBITION OF THIS PATHWAY RESULTS IN A SHIFT OF CELL DEATH TOWARDS NE CROTIC DEATH OF LESIONAL MACROPHAGES
(216) THEREBY AFFECTING LESION COMPOSITION 0REVIOUS BONE MARROW TRANSPLANTATION "-4 STUDIES DEMONSTRATED THAT P OF HEMATOPOIETIC ORIGIN CLEARLY HAS ATHEROPROTECTIVE PROPERTIES
(217) "ONE MARROW HARBORS NOT ONLY PROGENITORS FOR THE MYELOID LINEAGE INCLUDING MACROPHAGES
(218) BUT ALSO THE PROGENITORS FROM WHICH ULTIMATELY 4 CELLS AND " CELLS ORIGINATE )N ATHERO SCLEROSIS
(219) 4 CELL DERIVED CYTOKINES AS WELL AS " CELL MEDIATED ANTIBODY PRODUCTION CLEARLY CONTRIBUTE TO ATHEROSCLEROSIS PROGRESSION -OREOVER
(220) P IS SHOWN TO BE IMPORTANT IN 4 AND " CELL TURNOVER
(221) (ENCE
(222) THE ATHEROPROTECTIVE EFFECT OF BONE MARROW DERIVED P COULD BE ATTRIBUTED IN PART TO 4 AND " CELL SPECIlC P )N THIS LIGHT IT IS OF INTEREST THAT HEMATOPOIETIC P DElCIENCY IE COMBINED LYMPHOCYTE AND MACROPHAGE P DElCIENCY RESULTS IN STRONG EFFECTS ON THE SIZE OF THE LESIONS
(223) WHILE THIS STUDY DEMONSTRATES THAT MACROPHAGE RESTRICTED P DElCIENCY RESULTS TO MORE SUBTLE EFFECTS CONlNED TO LESIONAL MACROPHAGE TURNOVER
(224) EVENTUALLY AFFECTING LESION COMPOSITION #OMBINING THESE DATA IT CAN BE SUGGESTED THAT 4" CELL P IS IN VOLVED IN MODULATING SIZE IE GROWTH OF THE LESIONS &UTURE STUDIES
(225) USING CONDITIONAL APPROACHES FOR LYMPHOCYTE SPECIlC DELETION OF P MAY SHINE MORE LIGHT ONTO THE ROLE OF LYMPHOCYTE DERIVED P IN ATHEROGENESIS !DDITIONALLY
(226) THE DIFFERENCES IN lNDINGS ON QUANTITATIVE LESION AREA MAY ALSO PARTLY BE ATTRIBUTED TO THE DIFFERENCES IN CHOICE OF ATHEROSCLEROTIC BACKGROUND 7HEREAS THE ,$, RECEPTOR DElCIENT MOUSE MODEL AND THE !0/% ,EIDEN MOUSE MODEL SHOW MILD ATHEROSCLEROSIS DEVELOPMENT
(227) THE APO% MOUSE IS A MODEL OF AC CELERATED ATHEROSCLEROSIS DEVELOPMENT LEADING TO COMPLEX ATHEROSCLEROTIC LESIONS -OREOVER
(228) ABSENCE OF !PO% INHIBITS CHOLESTEROL EFmUX FROM MACROPHAGES AND THERE BY MORE PROGRESSIVELY STIMULATES THE FORMATION OF FOAM CELLS
(229) OUR CELLS OF INTEREST !LTHOUGH THE CLEARANCE OF APOPTOTIC CELL REMNANTS IS ATTENUATED IN APO% MICE THE FOAM CELL RICH LESIONS ENABLED US TO ESTABLISH THE ROLE OF MACROPHAGE P ON MACRO PHAGE TURNOVER IN THE ATHEROSCLEROTIC LESIONS "ASED ON THE CURRENT STUDY WE HYPOTHESIZE THAT IN EARLY LESIONS FOAM CELLS PREFERENTIALLY DIE QUICKLY VIA A RELATIVELY CLEAN APOPTOTIC DEATH WHICH REQUIRES P (OWEVER
(230) EVENTUALLY
(231) THE COMPOSITION OF THE LESIONS MAY PREVENT COMPLETE PROPER PHAGOCYTOSIS OF THE APOPTOTIC CELLS
(232) RESULTING IN SECONDARY NECROSIS AND THE FOR MATION OF A LIPID CORE )N CONTRAST
(233) UPON IMPAIRED FUNCTIONING OF THE P PATHWAY IN MACROPHAGES
(234) LESIONAL FOAM CELLS ARE NO LONGER DIRECTED TO DIE VIA THE APOPTOT IC PATHWAY 4HIS IS IN LINE WITH RECENT IN VITRO DATA OF -ERCER ET AL SHOWING THAT P PERITONEAL MACROPHAGES EXHIBIT REDUCED APOPTOSIS !S A CONSEQUENCE OF THE INABILITY TO DIE VIA APOPTOSIS
(235) DEATH OF FOAM CELLS MAY BE DELAYED
(236) BUT EVENTUALLY IT RE SULTS IN INCREASED DEATH VIA NECROSIS
(237) DUE TO EXTENSIVE LIPID ACCUMULATION 0REVIOUSLY
(238) IT WAS ALSO SHOWN THAT INHIBITION OF P IN MOUSE EMBRYONIC lBROBLASTS RESULTS IN A SHIFT OF ./ INDUCED CELL DEATH TOWARDS RELATIVELY MORE NECROSIS AND LESS APOPTOSIS .
(239) . -ACROPHAGE P AND ATHEROSCLEROSIS . 4HE ALTERNATIVE NECROTIC DEATH PATHWAY OF FOAM CELLS MAY BE MORE SLOWLY AND IS GEN ERALLY CONSIDERED TO BE MORE DETRIMENTAL SINCE NECROSIS LEADS TO THE RELEASE OF PRO INmAMMATORY AND PRO THROMBOTIC SUBSTANCES (ENCE
(240) MACROPHAGE NECROSIS IS DETRI MENTAL TO ATHEROSCLEROTIC LESION DEVELOPMENT
(241) WHEREAS MACROPHAGE APOPTOSIS CAN BE CONSIDERED MORE BENElCIAL IN LESION DEVELOPMENT AND PLAQUE STABILITY 4HEREFORE
(242) OUR DATA POINT TOWARDS AN IMPORTANT ROLE FOR MACROPHAGE P IN CONTROLLING FOAM CELL DEATH BY INDUCTION OF APOPTOSIS AND PREVENTION OF LESIONAL NECROSIS )N CONCLUSION
(243) WE DEMONSTRATE THAT MACROPHAGE P GUARANTEES SAFE FOAM CELL DEATH VIA APOPTOSIS AND THEREBY PREVENTS LESIONAL NECROSIS
(244) WHICH DIRECTLY AFFECTS LE SION COMPOSITION ,ESION COMPOSITION RATHER THAN LESION SIZE DETERMINES ITS VULNER ABILITY AND THEREBY ITS CLINICAL RELEVANCE 4HIS IMPLIES THAT LOCAL TARGETING OF PROCESS ES THAT REGULATE P MEDIATED APOPTOSIS MAY BE A POWERFUL TARGET FOR THERAPEUTIC INTERVENTION IN CORONARY ARTERY DISEASE 2ECENTLY
(245) THE USE OF DRUG ELUTING STENTS HAS EMERGED AS A HIGHLY PROMISING LOCAL APPROACH TO REDUCE IN STENT RESTENOSIS 4HE DIFFERENT DRUGS IE RAPAMYCIN
(246) mAVOPIRIDOL USED IN THESE DRUG ELUTING STENTS TARGET DIFFERENT APOPTOSIS AND PROLIFERATIVE GENES
(247) INCLUDING P $&.12:/('*(0(176 4HE AUTHORS WISH TO THANK $R * *ONKERS FOR HIS CONTRIBUTION TO THE EXPERIMENTS LEADING TO THE MANUSCRIPT 4HIS WORK WAS SUPPORTED BY THE .ETHERLANDS (EART &OUN DATION .(3 AND THE .ETHERLANDS /RGANIZATION FOR 3CIENTIlC 2ESEARCH .7/ "*- VAN 6LIJMEN IS A FELLOW OF THE 2OYAL .ETHERLANDS !CADEMY OF !RTS AND 3CIENCES
(248) AND -0* DE 7INTHER IS SUPPORTED BY .7/ AND 5()(5(1&(6 (ANSSON '+ )NmAMMATION
(249) ATHEROSCLEROSIS
(250) AND CORONARY ARTERY DISEASE . %NGL * -ED ,USIS !* !THEROSCLEROSIS .ATURE ,IBBY 0 #HANGING CONCEPTS OF ATHEROGENESIS * )NTERN -ED 2OSS 2 4HE PATHOGENESIS OF ATHEROSCLEROSIS A PERSPECTIVE FOR THE S .ATURE *)$ -EEK $7 4HE P RESPONSE TO $.! DAMAGE $.! 2EPAIR !MST +LEIN #
(251) 6ASSILEV ,4 4ARGETING THE P -$- INTERACTION TO TREAT CANCER "R * #ANCER 6OUSDEN +(
(252) ,U 8 ,IVE OR LET DIE THE CELLS RESPONSE TO P .AT 2EV #ANCER )HLING #
(253) -ENZEL '
(254) 7ELLENS %
(255) -ONTING *3
(256) 3CHAEFER (%
(257) :EIHER !- 4OPOGRAPHICAL ASSOCIATION BETWEEN THE CYCLIN DEPENDENT KINASES INHIBITOR 0
(258) P ACCUMULATION
(259) AND CELLULAR PROLIFERATION IN HUMAN ATHEROSCLEROTIC TISSUE !RTERIOSCLER 4HROMB 6ASC "IOL 'UEVARA .6
(260) +IM (3
(261) !NTONOVA %)
(262) #HAN , 4HE ABSENCE OF P ACCELERATES ATHEROSCLEROSIS BY INCREASING CELL PROLIFERATION IN VIVO .AT -ED VAN 6LIJMEN "*
(263) 'ERRITSEN '
(264) &RANKEN !,
(265) "OESTEN ,3
(266) +OCKX --
(267) 'IJBELS -*
(268) 6IERBOOM -0
(269) VAN %CK -
(270) VAN $E 7"
(271) VAN "ERKEL 4*
(272) (AVEKES ,- -ACROPHAGE P DElCIENCY LEADS TO ENHANCED ATHEROSCLEROSIS IN !0/% ,EIDEN TRANSGENIC MICE #IRC 2ES .
(273) #HAPTER . -ERCHED !*
(274) 7ILLIAMS %
(275) #HAN , -ACROPHAGE SPECIlC P EXPRESSION PLAYS A CRUCIAL ROLE IN ATHEROSCLEROSIS DEVELOPMENT AND PLAQUE REMODELING !RTERIOSCLER 4HROMB 6ASC "IOL *ONKERS *
(276) -EUWISSEN 2
(277) VAN $ER '(
(278) 0ETERSE (
(279) VAN D
(280) 6
(281) "ERNS ! 3YNERGISTIC TUMOR SUPPRESSOR ACTIVITY OF "2#! AND P IN A CONDITIONAL MOUSE MODEL FOR BREAST CANCER .AT 'ENET #LAUSEN "%
(282) "URKHARDT #
(283) 2EITH 7
(284) 2ENKAWITZ 2
(285) &ORSTER ) #ONDITIONAL GENE TARGETING IN MACROPHAGES AND GRANULOCYTES USING ,YS-CRE MICE 4RANSGENIC 2ES :HANG 3(
(286) 2EDDICK 2,
(287) 0IEDRAHITA *!
(288) -AEDA . 3PONTANEOUS HYPERCHOLESTEROLEMIA AND ARTERIAL LESIONS IN MICE LACKING APOLIPOPROTEIN % 3CIENCE .ISHINA 0-
(289) 6ERSTUYFT *
(290) 0AIGEN " 3YNTHETIC LOW AND HIGH FAT DIETS FOR THE STUDY OF ATHEROSCLEROSIS IN THE MOUSE * ,IPID 2ES -EULMEESTER %
(291) -AURICE --
(292) "OUTELL #
(293) 4EUNISSE !&
(294) /VAA (
(295) !BRAHAM 4%
(296) $IRKS 27
(297) *OCHEMSEN !' ,OSS OF (!530 MEDIATED DEUBIQUITINATION CONTRIBUTES TO $.! DAMAGE INDUCED DESTABILIZATION OF (DMX AND (DM -OL #ELL "OESTEN ,3
(298) :ADELAAR !3
(299) VAN .IEUWKOOP !
(300) 'IJBELS -*
(301) DE 7INTHER -0
(302) (AVEKES ,-
(303) VAN 6LIJMEN "* 4UMOR NECROSIS FACTOR ALPHA PROMOTES ATHEROSCLEROTIC LESION PROGRESSION IN !0/% LEIDEN TRANSGENIC MICE #ARDIOVASC 2ES 0AIGEN "
(304) -ORROW !
(305) (OLMES 0!
(306) -ITCHELL $
(307) 7ILLIAMS 2! 1UANTITATIVE ASSESSMENT OF ATHEROSCLEROTIC LESIONS IN MICE !THEROSCLEROSIS 4ANGIRALA 2+
(308) 2UBIN %-
(309) 0ALINSKI 7 1UANTITATION OF ATHEROSCLEROSIS IN MURINE MODELS CORRELATION BETWEEN LESIONS IN THE AORTIC ORIGIN AND IN THE ENTIRE AORTA
(310) AND DIFFERENCES IN THE EXTENT OF LESIONS BETWEEN SEXES IN ,$, RECEPTOR DElCIENT AND APOLIPOPROTEIN % DElCIENT MICE * ,IPID 2ES +ANTERS %
(311) 0ASPARAKIS -
(312) 'IJBELS -*
(313) 6ERGOUWE -.
(314) 0ARTOUNS (ENDRIKS )
(315) &IJNEMAN 2*
(316) #LAUSEN "%
(317) &ORSTER )
(318) +OCKX --
(319) 2AJEWSKY +
(320) +RAAL '
(321) (OFKER -(
(322) DE 7INTHER -0 )NHIBITION OF .& KAPPA" ACTIVATION IN MACROPHAGES INCREASES ATHEROSCLEROSIS IN ,$, RECEPTOR DElCIENT MICE * #LIN )NVEST $AMALAS !
(323) "EN :E%V !
(324) 3IMCHA )
(325) 3HTUTMAN -
(326) ,EAL *&
(327) :HURINSKY *
(328) 'EIGER "
(329) /REN - %XCESS BETA CATENIN PROMOTES ACCUMULATION OF TRANSCRIPTIONALLY ACTIVE P %-"/ * -OLLER -"
(330) 'ERDES !-
(331) 3KJODT +
(332) -ORTENSEN ,3
(333) 0EDERSEN .4 $ISRUPTED P FUNCTION AS PREDICTOR OF TREATMENT FAILURE AND POOR PROGNOSIS IN " AND 4 CELL NON (ODGKINS LYMPHOMA #LIN #ANCER 2ES 7ARD *-
(334) 4ADESSE (EATH ,
(335) 0ERKINS 3.
(336) #HATTOPADHYAY 3+
(337) (URSTING 3$
(338) -ORSE (#
(339) ))) 3PLENIC MARGINAL ZONE " CELL AND THYMIC 4 CELL LYMPHOMAS IN P DElCIENT MICE ,AB )NVEST 2OSENFELD -%
(340) 0OLINSKY 0
(341) 6IRMANI 2
(342) +AUSER +
(343) 2UBANYI '
(344) 3CHWARTZ 3- !DVANCED ATHEROSCLEROTIC LESIONS IN THE INNOMINATE ARTERY OF THE !PO% KNOCKOUT MOUSE !RTERIOSCLER 4HROMB 6ASC "IOL -EIR +3
(345) ,EITERSDORF % !THEROSCLEROSIS IN THE APOLIPOPROTEIN % DElCIENT MOUSE A DECADE OF PROGRESS !RTERIOSCLER 4HROMB 6ASC "IOL 'RAINGER $*
(346) 2ECKLESS *
(347) -C+ILLIGIN % !POLIPOPROTEIN % MODULATES CLEARANCE OF APOPTOTIC BODIES IN VITRO AND IN VIVO
(348) RESULTING IN A SYSTEMIC PROINmAMMATORY STATE IN APOLIPOPROTEIN % DElCIENT MICE * )MMUNOL 4ABAS ) P AND ATHEROSCLEROSIS #IRC 2ES -ERCER *
(349) &IGG .
(350) 3TONEMAN 6
(351) "RAGANZA $
(352) "ENNETT -2 %NDOGENOUS P PROTECTS VASCULAR SMOOTH MUSCLE CELLS FROM APOPTOSIS AND REDUCES ATHEROSCLEROSIS IN !PO% KNOCKOUT MICE #IRC 2ES -C,AUGHLIN ,-
(353) $EMPLE " .ITRIC OXIDE INDUCED APOPTOSIS IN LYMPHOBLASTOID AND lBROBLAST CELLS DEPENDENT ON THE PHOSPHORYLATION AND ACTIVATION OF P #ANCER 2ES VAN DER (OEVEN ",
(354) 0IRES .-
(355) 7ARDA (-
(356) /EMRAWSINGH 06
(357) VAN 6LIJMEN "*
(358) 1UAX 0(
(359) 3CHALIJ -*
(360) VAN DER 7ALL %%
(361) *UKEMA *7 $RUG ELUTING STENTS RESULTS
(362) PROMISES AND PROBLEMS )NT * #ARDIOL .
(363)
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