Cover Page
The handle http://hdl.handle.net/1887/19855 holds various files of this Leiden University dissertation.
Author: Solingen, Coen van
Title: The role of microRNA-126 in vascular homeostasis Date: 2012-09-26
',%48)6
1MGVS62%QSHYPEXIWIRHSXLIPMEP
7(*I\TVIWWMSRERHQSFMPM^EXMSRSJ
7GE
0MR
TVSKIRMXSVGIPPWMRMWGLIQME
'SIRZER7SPMRKIR,IXX]'HI&SIV6SIP&MNOIVO1EXXLMIY1SRKI
%RRIQEVMI1ZER3IZIVIR6MIXHMNO0ISREVH7IKLIVW 1EVKVIIX6HI:VMIW)VMG4ZERHIV:IIV4EYP,%5YE\
8SR.6EFIPMROERH%RXSR.ERZER>SRRIZIPH
(ITEVXQIRXSJ2ITLVSPSK]ERHXLI)MRXLSZIR0EFSVEXSV]JSV)\TIVMQIRXEP
:EWGYPEV1IHMGMRI(ITEVXQIRXSJ7YVKIV]091'0IMHIRXLI2IXLIVPERHW
'EVHMSZEWG6IW(IG
%FWXVEGX
1MGVS62%QM6 [LMGLMWIRVMGLIHMRIRHSXLIPMEPGIPPWTPE]WEVSPI
MRERKMSKIRIWMW&EWIHSRXLIWIIHWIUYIRGIQM6GEREPWSFITVIHMGXIHXS
VIKYPEXIZEWGYPSKIRIWMWF]QSHYPEXMRKXLIIRHSXLIPMEPI\TVIWWMSRSJWXVSQEPGIPP
HIVMZIHJEGXSV7(*
9WMRK QM6VITSVXIV GSRWXVYGXW [I ½VWX ZEPMHEXIH XLEX QM6
MRLMFMXW 7(* I\TVIWWMSR MR IRHSXLIPMEP GIPPW MR ZMXVS 2I\X [I MRZIWXMKEXIH XLI TSXIRXMEP VIPIZERGI SJ XLMW SFWIVZEXMSR [MXL VIWTIGX XS XLI
QSFMPM^EXMSR SJ TVSKIRMXSV GIPPW *SV XLMW [I WXYHMIH XLI QMKVEXMSR SJ LYQER
'(TVSKIRMXSVGIPPWXS[EVHWGLIQSXEGXMGJEGXSVWTVIWIRXMRIRHSXLIPMEPGIPP
GSRHMXMSRIH QIHMYQ%RXEKSQMVMRHYGIH WMPIRGMRK SJ QM6 IPIZEXIH 7(*
I\TVIWWMSRF]LYQERYQFMPMGEPZIMRIRHSXLIPMEPGIPPWERHIRLERGIHQMKVEXMSRSJ
XLI'(GIPPW-REQYVMRIQSHIPSJLMRHPMQFMWGLIQMEEWXVMOMRKMRGVIEWIMR
XLIRYQFIVSJGMVGYPEXMRK7GE0MRTVSKIRMXSVGIPPWMRERXEKSQMVXVIEXIH
QMGI [EW SFWIVZIH EW GSQTEVIH XS WGVEQFPIQMVXVIEXIH GSRXVSPW -QQYRS
LMWXSGLIQMGEPWXEMRMRKSJGETMPPEVMIWMRXLITSWXMWGLIQMGKEWXVSGRIQMYWQYWGPI
SJ QM6WMPIRGIH QMGI VIZIEPIH IPIZEXIH 7(* I\TVIWWMRK '(TSWMXMZI
GETMPPEVMIW[LIVIEWEQSFMPM^MRKIJJIGXSJQM6MRLMFMXMSR[EWRSXHIXIGXIHMR
LIEPXL]GSRXVSPERMQEPW
QM6 GER VIKYPEXI XLI I\TVIWWMSR SJ 7(* MR IRHSXLIPMEP GIPPW -R XLI
GSRXI\X SJ ER MWGLIQMG IZIRX W]WXIQMG WMPIRGMRK SJ QM6 PIEHW XS XLI
QSFMPM^EXMSR SJ 7GE0MR TVSKIRMXSV GIPPW MRXS XLI TIVMTLIVEP GMVGYPEXMSR
TSXIRXMEPP]MRVIWTSRWIXSIPIZEXIH7(*I\TVIWWMSRF]IRHSXLIPMEPGIPPWTVIWIRX
MRXLIMWGLIQMGXMWWYI
-RXVSHYGXMSR
-RXIKVMX]SJXLIZEWGYPEVIRHSXLIPMYQMWGIRXVEPXSZEWGYPEVLSQISWXEWMWERH
MWHIXIVQMRIHF]XLIFEPERGIFIX[IIRIRHSXLIPMEPMRNYV]ERHVITEMV?A-RXLI
GSRXI\X SJ EHZIVWI LIQSH]REQMG ERH QIXEFSPMG VMWO JEGXSVW IRHSXLIPMEP GIPPW
)'W EVI HEQEKIH ERH GSRWXERXP] VITPEGIH F] XLI TVSPMJIVEXMSR SJ EHNEGIRX
QEXYVI)'W-RXLIVEXESVXE)'WMREVIEWVIWMWXERXXSEXLIVSWGPIVSWMWLEZIEPS[
VEXISJGIPPYPEVVITPMGEXMSRERHLEZIFIIRIWXMQEXIHXSLEZIEPMJIWTERSJETTVS\M
QEXIP]QSRXLW-RGSRXVEWX)'XYVRSZIVMRPIWMSRTVSRIWMXIWMWEGGIPIVEXIH
XS[IIOWSVPIWWEWERMQEPWEKI?A8SQEMRXEMRXLIMRXIKVMX]SJXLIIRHSXLIPMYQ
XLVSYKLSYX PMJI IRHSXLIPMEP GIPPW MR XLI EVXIVMEP [EPP [SYPH XLIVIJSVI LEZI XS
VITPMGEXI[IPPSZIVEXLSYWERHXMQIW,S[IZIVTVMQEV]IRHSXLIPMEPGIPPGYPXYVIW
EPVIEH]FIGSQIWIRIWGIRXEJXIVEPMQMXIHRYQFIVSJTEWWEKIWHYIXSETVSKVIWWMZI
WLSVXIRMRK SJ XIPSQIVIW XLEX XEOIW TPEGI YTSR IEGL GIPP HMZMWMSR 1SVISZIV
XIPSQIVI IVSWMSR MW EGGIPIVEXIH F] GLVSRMG I\TSWYVI SJ IRHSXLIPMEP GIPPW XS
S\MHEXMZI WXVIWW ?A 'SRWIUYIRXP] MX LEW FIIR WYKKIWXIH XLEX QEXYVI )'W
EVI MRWYJ½GMIRXP] GETEFPI SJ VITEMVMRK XLI GLVSRMGEPP] HEQEKIH IRHSXLIPMYQ
XLVSYKLSYX PMJI 7YTTSVX JSV XLMW RSXMSR LEW FIIR TVSZMHIH F] WIZIVEP
WXYHMIWMHIRXMJ]MRKEWMKRM½GERXGSRXVMFYXMSRF]IRHSXLIPMEPTVSKIRMXSVGIPPWEWER
EPXIVREXMZIGIPPYPEVWSYVGIJSVVIIRHSXLIPMEPM^EXMSRSJXLIHEQEKIHEVXIV][EPP
?A
7XVSQEP GIPP HIVMZIH JEGXSV 7(* SV '<'0 LEW FIIR HIQSRWXVEXIH
XS JEGMPMXEXI XLI LSQMRK SJ TVSKIRMXSV GIPPW JVSQ XLI TIVMTLIVEP GMVGYPEXMSR XS
WMXIW SJ ZEWGYPEV MRNYV] SV XMWWYI MWGLIQME 7(* GER FI EGXMZIP] WIGVIXIH F]
MRNYVIHIRHSXLIPMEPGIPPW?ASVEGXMZEXIHTPEXIPIXW?APIEHMRKXSXLILSQMRKSJFSRI
QEVVS[QSFMPM^IH TVSKIRMXSV GIPPW XS XLI WMXI SJ MRNYV] ? A8LI FIRI½GMEP
VSPI SJ 7(* MR VIGVYMXMRK FSRI QEVVS[ GIPPW XS MWGLIQMG XMWWYI LEW FIIR
IWXEFPMWLIHMRWIZIVEPERMQEPQSHIPWXSMQTVSZIVIGSZIV]EJXIVERMWGLIQMGIZIRX
?A8LIVIJSVI7(*MWFIPMIZIHXSTPE]EGIRXVEPVSPIMRVIKYPEXMRKFSXL
ZEWGYPEVMRXIKVMX]ERHLSQISWXEWMWMQTPMGEXMRKXLEXMXWI\TVIWWMSRWLSYPHFIXMKLXP]
GSRXVSPPIH -RHIIH 7(* I\TVIWWMSR MW VITSVXIH XS FI VIKYPEXIH EX QYPXMTPI
PIZIPWMRGPYHMRKXVERWGVMTXMSRERHTSWXXVERWPEXMSR?A
%X XLI TSWXXVERWGVMTXMSREP PIZIP 7(* I\TVIWWMSR GSYPH FI VIKYPEXIH F]
QMGVS62%WQM62%W 1M62%WGSRWXMXYXIEGPEWWSJLMKLP]GSRWIVZIHRSRGSHMRK
62%WXLEXGSRXVSPKIRII\TVIWWMSRF]MRLMFMXMRKXLIXVERWPEXMSRSJQ62%?A
8LI EFMPMX] SJ QM62%W XS VIKYPEXI QYPXMTPI XEVKIXW TVSZMHIW E QIERW JSV XLI
GSSVHMREXIH GSRXVSP SJ KIRI I\TVIWWMSR ERH QEOI XLIWI QSPIGYPIW IWTIGMEPP]
EXXVEGXMZIGERHMHEXIWJSVVIKYPEXMRKGIPPX]TIWTIGM½GHMJJIVIRXMEXMSRERHQSHYPEXMRK
GIPPJYRGXMSR?A-RVIGIRX]IEVWQM62%I\TVIWWMSRTVS½PIWSJ)'WLEZIFIIR
EREP]^IHMRHIXEMPERHVIGIRXWXYHMIWHIQSRWXVEXIHFSXLTVSERKMSKIRMG?A
EW[IPPEWERXMERKMSKIRMGJYRGXMSRWJSVIRHSXLIPMEPQM62%W?A
-R WMPMGS EREP]WIW LXXT[[[QMGVSVRESVK JSV QM62%W XEVKIXMRK XLI ´
YRXVERWPEXIH VIKMSR ´ 986 SJ XLI 7(* Q62% MHIRXM½IH QM6 EW E
TSXIRXMEPTSWXXVERWGVMTXMSREPVIKYPEXSVSJ7(*I\TVIWWMSR1M6MWLMKLP]
IRVMGLIH MR )'W ERH LEW FIIR HIQSRWXVEXIH XS VIKYPEXI MWGLIQMEMRHYGIH ERKMSKIRIWMWF]FPSGOMRKXLII\TVIWWMSRSJ746)(ERH4-/6?A,IVI
[IZEPMHEXIHXLIMRLMFMXSV]EGXMSRWSJQM6SRI\TVIWWMSRERHJYRGXMSRSJ
7(*MR)'WMRZMXVS*YVXLIVQSVI[IHIQSRWXVEXIXLEXERXEKSQMVMRHYGIHWM
PIRGMRKSJQM6TVMSVXSEGYXIMWGLIQMEPIEHWXSXLIEYKQIRXEXMSRSJ7(*
PIZIPW MR FSXL XLI GMVGYPEXMSR ERH MWGLIQMG XMWWYI 8LMW MW EWWSGMEXIH [MXL
MRGVIEWIH QSFMPM^EXMSR SJ FSRI QEVVS[HIVMZIH TVSKIRMXSV GIPPW MR ZMZS 3YV
HEXE HIQSRWXVEXI XLEX IRHSXLIPMEP QM6 GER VIKYPEXI FSXL JIEXYVIW SJ
ERKMSKIRIWMW EW [IPP EW ZEWGYPSKIRIWMW ERH WYKKIWX E VSPI JSV QM6 MR XLI
QEMRXIRERGISJIRHSXLIPMEPLSQISWXEWMW
1EXIVMEPERHQIXLSHW
'IPPWERHGIPPGYPXYVI
-QQSVXEPM^IH LYQER YQFMPMGEP ZIMR IRHSXLIPMEP GIPPW )'6* ?A [IVI
GYPXYVIH MR 1 QIHMYQ +MFGS &VIHE XLI 2IXLIVPERHW WYTTPIQIRXIH
[MXL TIRMGMPPMRWXVITXSQ]GMR +MFGS-RZMXVSKIR JIXEP GEPJ WIVYQ *'7 &MS
;LMXXEOIV'EQFVI\:IVZMIVW&IPKMYQ -9Q0LITEVMR0IS4LEVQE&VIHE
XLI 2IXLIVPERHW ,)4)7 FYJJIV +MFGS ERH ÜKQ0 IRHSXLIPMEP
GIPP KVS[XL WYTTPIQIRX 7MKQE%PHVMGL 7X 0SYMW 13 ,YQER YQFMPMGEP ZIMR
IRHSXLIPMEPGIPPW,9:)' [IVIMWSPEXIHGYPXYVIHERHGLEVEGXIVM^IHEWHIWGVMFIH
TVIZMSYWP]?A-RWLSVX)'W[IVIMWSPEXIHJVSQJVIWLP]SFXEMRIHLYQERYQFMPMGEP
GSVHWF]XV]TWMR)(8%HMKIWXMSRSJXLIMRXIVMSVSJXLIYQFMPMGEPZIMR8LIGIPPW
[IVIGYPXYVIHMR1QIHMYQWYTTPIQIRXIH[MXLTIRMGMPPMRWXVITXSQ]GMR
*'7 -9Q0 LITEVMR ERH FSZMRI TMXYMXEV] I\XVEGX +MFGS '( GIPPW
[IVIMWSPEXIHJVSQYQFMPMGEPGSVHFPSSHYWMRK*MGSPP%QIVWLEQ³W,IVXSKIR
FSWGL XLI 2IXLIVPERHW HIRWMX] KVEHMIRX GIRXVMJYKEXMSR ERH TSWMXMZI WIPIGXMSR
YWMRK'(1%'7QMGVSFIEHW1MPXIR]M&MSXIGL&IVKMWL+PEHFEGL+IVQER]
9QFMPMGEP GSVH FPSSH ERH YQFMPMGEP GSVHW [IVI GSPPIGXIH RSRMHIRXM½EFPI ERH
[MXL MRJSVQIH GSRWIRX 7MRGI XLI LYQER XMWWYIW [IVI YRMHIRXM½EFPI ETTVSZEP
JVSQXLIYRMZIVWMX]IXLMGWVIZMI[FSEVH[EWRSXRIGIWWEV]
7(*´986VITSVXIVEWWE]W
7]RXLIXMGHSYFPIWXVERHIHSPMKSRYGPISXMHIWWTERRMRKEFEWITEMVVIKMSRSJ
XLIQYVMRI´986SJ7(*Q62%GSRXEMRMRKXLITYXEXMZIQM6FMRHMRKWMXI
[MXLT7(*QQ SV[MXLSYXT7(* E'XS+QMWQEXGLEXTSWMXMSRSJXLIWIIH
WIUYIRGI [IVI GPSRIH MRXS XLI T1-6VITSVX81 )\TVIWWMSR 6ITSVXIV:IGXSV
7]WXIQ %TTPMIH &MSW]WXIQW%QWXIVHEQ XLI 2IXLIVPERHW WII WYTTPIQIRXEV]
½PIJSVSPMKSRYGPISXMHIWIUYIRGIW %PPTPEWQMHW[IVIWIUYIRGIHXSGSR½VQXLIMV
WXVYGXYVIERHI\GPYHIGPSRMRKEVXIJEGXW%VIRMPPEPYGMJIVEWII\TVIWWMSRTPEWQMH
T607:4VSQIKE0IMHIRXLI2IXLIVPERHW ERHETPEWQMHGSRXEMRMRKEWMRKPI
TIVJIGXQEXGLQM6FMRHMRKWMXIWIVZIHEWGSRXVSPW?A
(IWMKRSJERXEKSQMVW
%RXEKSQMV ERH E GSRXVSP WGVEQFPIQMV (LEVQEGSR 62% XIGLRSPSKMIW
0EJE]IXXI'3 [IVIW]RXLIWM^IHEWTVIZMSYWP]HIWGVMFIH?A
0YGMJIVEWIEWWE]
%RXEKSQMV SV GSRXVSP WGVEQFPIQMV ÜKQ0 [EW EHHIH XS E RIEV
GSR¾YIRXPE]IVSJ½FVSRIGXMREHLIVIRX)'6*GIPPW8[IRX]JSYVLSYVWEJXIV
ERXEKSQMV XVIEXQIRX XLI )'6* GIPPW [IVI HIXEGLIH F] XV]TWMRM^EXMSR ERH
VIWYWTIRHIHMRÜ0WIVYQJVII3TXMQIQGYPXYVIQIHMYQ+MFGS ERH
ÜKWTIGM½GT1-6VITSVXERHRKT607:[IVIEHHIH8LIGIPPWYWTIRWMSR
[EWGLMPPIHJSVQMRYXIWEX'ERHIPIGXVSTSVEXIHYWMRKXLI+IRI4YPWIV--
&MS6EH0EFSVEXSVMIW:IIRIRHEEPXLI2IXLIVPERHW %JXIVQMRYXIWVIGSZIV]
XMQIEXVSSQXIQTIVEXYVI\GIPPW[IVITPEXIHMRE[IPPWTPEXIGSEXIH
[MXL ½FVSRIGXMR MR XVMTPMGEXI%JXIV LSYVW XLI ½VI¾]PYGMJIVEWI ERH VIRMPPE
PYGMJIVEWI WMKREPW [IVI QIEWYVIH YWMRK E (YEP0YGMJIVEWI %WWE] 6ITSVXIV 7]WXIQ4VSQIKE MRE0YQEX0&PYQMRSQIXIV)++&IVXLSPH&YRHSSVE
%YWXVEPME
;IWXIVRFPSX
,9:)' [IVI MRGYFEXIH JSV LSYVW [MXL ÜKQ0 ERXEKSQMV SV
WGVEQFPIQMV'YPXYVIQIHMYQ[EWEWTMVEXIHERHGIPPW[IVI[EWLIHX[SXMQIW
[MXL 4&7 GIPPYPEV P]WEXIW [IVI LEVZIWXIH YWMRK P]WMW FYJJIV Q18VMW,'P
T,! Q1 2E'P 7(7 WSHMYQHIS\]GLSPEXI ERH 8VMXSR
<7MKQE%PHVMGL 4VSXIMRPIZIPWJSV7(*ERHơEGXMR[IVIEWWIWWIHF]
;IWXIVR FPSX EREP]WMW [MXL GLIQMPYQMRIWGIRGI HIXIGXMSR )UYEP EQSYRXW SJ
TVSXIMR[IVIVIWSPZIHSR 7(7TSP]EGV]PEQMHIKIPWERHXVERWJIVVIHXS4:(*
QIQFVERIW 1MPPMTSVI &MPPIVMGE 1% 7(* [EW HIXIGXIH YWMRK E TSP]GPSREP
VEFFMXERXMFSH]EKEMRWXLYQER7(*YKQ0%FGEQ'EQFVMHKI9/ ERHJSV
ơEGXMRETSP]GPSREPVEFFMXERXMFSH]EKEMRWXLYQERơEGXMRYKQ0%FGEQ [EWYWIH,SVWIVEHMWLTIVS\MHEWIGSRNYKEXIHKSEXERXMVEFFMX-K+YKQ0
(EOS']XSQEXMSR)RWGLIHIXLI2IXLIVPERHW [EWYWIHEWWIGSRHEV]ERXMFSH]
&SYRH JVEKQIRXW [IVI HIXIGXIH [MXL GLIQMPYQMRIWGIRX VIEKIRXW 7YTIVWMKREP
;IWX (YVE )\XIRHIH (YVEXMSR 7YFWXVEXI8LIVQSWGMIRXM½G ERH I\TSWIH SR
,]TIV½PQ)'0%QIVWLEQ 5YERXMXEXMZIEREP]WMWSJXLI7(*FERHMRXIRWMX]SR
;IWXIVRFPSX[EWTIVJSVQIHYWMRKMQEKI.WSJX[EVIERHRSVQEPM^IHJSVơEGXMR
8LI VEXMS SJ XLI 7(* FERH MRXIRWMX] SZIV XLI ơEGXMR FERH MRXIRWMX] SJ XLI ERXEKSQMVXVIEXIH,9:)'[EWEVFMXVEVMP]WIXEX
1MKVEXMSREWWE]
,9:)'[IVIMRGYFEXIHMRWIVYQJVIIGIPPWTIGM½GQIHMYQ[MXL MRWYPMR
XVERWJIVVMRWSHMYQ WIPIRMXI QIHME WYTTPIQIRX -87 7MKQE%PHVMGL GSRXEMRMRK
ERXEKSQMV SV WGVEQFPIQMV ÜKQ0 EX '%JXIV LSYVW GIPP WYTIV
REXERXW[IVILEVZIWXIHERHTPEGIHMRXSXLIPS[IVGSQTEVXQIRXSJEXVERW[IPP
W]WXIQ [MXL E TSVI WM^I SJ ÜQ 'SVRMRK &: 0MJI 7GMIRGIW%QWXIVHEQ XLI
2IXLIVPERHW ,YQER '( GIPPW [IVI EHHIH XS XLI YTTIV GLEQFIV ERH QMKVEXMSR[EWJSPPS[IHSZIVETIVMSHSJLSYVWEX'%WETSWMXMZIGSRXVSP
RKQ0 VIGSQFMRERX LYQER 7(*6( 7]WXIQW [EW EHHIH MR XLI PS[IV
GSQTEVXQIRX8LI 7(*VIGITXSV '<'6 [EW RIYXVEPM^IH F] MRGYFEXMSR SJ
'(GIPPW[MXLEFPSGOMRKERXMFSH]EKEMRWX'<'6ÜKQ06(7]WXIQW JSVQMRYXIWEX'%JXIVLXLIGIPPWYWTIRWMSRWMRXLIPS[IVGSQTEVXQIRX
[IVILEVZIWXIHWTYRHS[RERHMRGYFEXIHJSVQMRYXIWEX'MR*%'7FYJJ
IV4&7 FSZMRIWIVYQEPFYQMR&7%7MKQE%PHVMGL [MXLHMVIGXP]GSRNY
KEXIHERXMFSHMIWEKEMRWXLYQER'(4IV'4']&(&MSWGMIRGIW&VIHEXLI
2IXLIVPERHW ERH LYQER '( %4' 1MPXIR]M &MSXIG % ½\IH RYQFIV SJ
'( GIPPW [EW ¾YSVIWGIRXP] PEFIPIH F] MRGYFEXMSR [MXL GEPGIMR%1 6(
7]WXIQW1MRRIETSPMW1( ERHEHHIHTVMSVXS*%'7EREP]WMW
,MRHPMQFMWGLIQMEQSHIP
%PP ERMQEP I\TIVMQIRXEP TVSXSGSPW [IVI ETTVSZIH F] XLI ERMQEP [IPJEVI
GSQQMXXII SJ XLI 2IXLIVPERHW 3VKERM^EXMSR JSV %TTPMIH 7GMIRXM½G 6IWIEVGL
8230IMHIRXLI2IXLIVPERHW ERHGSRJSVQXLI(MVIGXMZI)9SJXLI
)YVSTIER 4EVPMEQIRX3RI HE] TVMSV XS WYVKIV] '&0;8 QMGI R! TIV
KVSYTEKI![IIOW'LEVPIW6MZIV1EEWXVMGLXXLI2IXLIVPERHW [IVIMRNIGXIH
MRXVEZIRSYWP]Ü0 [MXLERXEKSQMVQKERMQEP SVWGVEQFPIQMV
QKERMQEP &IJSVI WYVKIV] QMGI [IVI ERIWXLIXM^IH MRXVETIVMXSRIEPP] [MXL E
GSQFMREXMSR SJ 1MHE^SPEQ QKOK 6SGLI (MEKRSWXMGW %PQIVI XLI
2IXLIVPERHW 1IHIXSQMHMRIQKOK3VMSR'SVTSVEXMSR8YVOY*MRPERH ERH
*IRXER]PQKOK.ERWWIR4LEVQEGIYXMGE8MPFYVKXLI2IXLIVPERHW -WGLIQME
SJ XLI PIJX LMRH PMQF [EW MRHYGIH F] IPIGXVSGSEKYPEXMSR SJ XLI PIJX GSQQSR
JIQSVEP EVXIV] TVS\MQEP XS XLI FMJYVGEXMSR SJ WYTIV½GMEP ERH HIIT JIQSVEP
EVXIV]EWHIWGVMFIH?A8SWEGVM½GIXLIQMGIFPSSH[EW[MXLHVE[RJSV*%'7
EREP]WMW )(8%ERXMGSEKYPEXIH F] LIEVX TYRGXYVI ERH XLI XMFME ERH JIQYV
SJ FSXL PIKW [IVI OITX JSV MWSPEXMSR SJ FSRI QEVVS[ GIPPW%HHMXMSREPP] XLI
KEWXVSGRIQMYW QYWGPI SJ FSXL LMRH PMQFW [EW HMWWIGXIH ERH TPEGIH MR JSVQEPHIL]HI SZIVRMKLX %JXIV TEVEJ½R IQFIHHMRK ÜQ XLMGO WIVMEP GVSWW
WIGXMSRW[IVIQEHIJSVMQQYRSLMWXSGLIQMGEPEREP]WMW
;LSPIFPSSHEREP]WMW
;LSPI FPSSH [EW GSPPIGXIH F] MRGMWMSR SJ XLI XEMP ZIMR SV LIEVX TYRGXYVI
ERH EREP]^IH F] WIQMEYXSQEXMG LIQEXSPSK] EREP]^IV * 7]WQI\ 7]WQI\
'SVTSVEXMSR )XXIR0IYV XLI 2IXLIVPERHW *%'7EREP]WMW SV )0-7% ,IQEXS
PSKMGEP ZEPYIW SFXEMRIH [IVI [LMXI FPSSH GIPP GSYRXW ;&' RQ0 VIH
FPSSHGIPPGSYRXW6&'RQ0 TPEXIPIXW408RQ0 LIQEXSGVMX,'8
ERHLEIQSKPSFMR,+&QQSP0 *SV*%'7EREP]WMW[IMRGYFEXIHÜ0SJ
[LSPIFPSSHJSVQMRYXIWEX'[MXLHMVIGXP]GSRNYKEXIHERXMFSHMIWHMVIGXIH
EKEMRWX7GE*-8'&(&MSWGMIRGIW *PO4)&(4LEVQMRKIR7ER(MIKS'% ERHEGSGOXEMPEKEMRWXPMRIEKITSWMXMZIGIPPW%4'&(4LEVQMRKIR -REWITEVEXI
XYFIÜ0SJ[LSPIFPSSH[EWMRGYFEXIH[MXLERETTVSTVMEXIGSGOXEMPSJMWSX]TI
GSRXVSPWXSMHIRXMJ]XLIXLVIWLSPHJSVPMRIEKITSWMXMZMX]7(*PIZIPWMRWIVYQ
[IVIEWWIWWIH[MXL)0-7%6(7]WXIQW
-QQYRSLMWXSGLIQMWXV]-,'
*SYV ÜQXLMGO WIGXMSRW SJ QYVMRI KEWXVSGRIQMYW QYWGPI [IVI VIL]HVEXIH
ERH MRGYFEXIH [MXL ERXMKIRVIXVMIZEP FYJJIV XV]TWMR)(8% MR 4&7 2I\X
WIGXMSRW[IVIMRGYFEXIH[MXLWTIGM½GERXMFSHMIWEKEMRWXQYVMRI7(*VEFFMX
TSP]GPSREP -K+ 8LIVQSWGMIRXM½G 6SGOJSVH -0 ERH QYVMRI '( QSYWI
QSRSGPSREP -K+F 7ERXE 'VY^ ,IMHIPFIVK +IVQER] JSPPS[IH F] WIGSRHEV]
ERXMFSHMIW EKEMRWX KSEXERXMVEFFMX-K+ PEFIPIH [MXL %PI\E SV KSEXERXM
QSYWI -K+ PEFIPIH [MXL%PI\E 1SPIGYPEV 4VSFIW %W E RIKEXMZI GSRXVSP
MWSX]TIQEXGLIH -K+W [IVI YWIH -QEKIW [IVI QEHI YWMRK E GSRJSGEP
QMGVSWGSTI'EVP>IMWW7PMIHVIGLXXLI2IXLIVPERHW
*%'7EREP]WMW
%PP WEQTPIW SFXEMRIH JSV *%'7 EREP]WMW [IVI IMXLIV MQQIHMEXIP] EREP]^IH
F]¾S[G]XSQIXV]EREP]WMW*%'7076--&(&MSWGMIRGIW SV[IVI½\IHMR TEVEJSVQEPHIL]HI ERH EREP]^IH [MXLMR LSYVW EJXIV TVITEVEXMSR (EXE [IVI
EREP]^IHYWMRK*%'7(M:EWSJX[EVI&(&MSWGMIRGIW 7XEXMWXMGEPEREP]WMW
6IWYPXWEVII\TVIWWIHEWWXERHEVHIVVSVSJXLIQIER7)1 7XEXMWXMGEPEREP]WMW
[EW TIVJSVQIH YWMRK XLI 1ERR;LMXRI] 8XIWX 4 [EW GSRWMHIVIH
WXEXMWXMGEPP]WMKRM½GERX
6IWYPXW
1M6EJJIGXWXLII\TVIWWMSRERHJYRGXMSRSJ7(*MRZMXVS 9WMRK ER SRPMRI QM62% XEVKIX WIEVGL XSSP LXXT [[[QMGVSVRESVK [I
MHIRXM½IHETYXEXMZIQM6FMRHMRKWMXIMRXLI´986SJXLI7(*Q62%8S
EWWIWWMXWJYRGXMSREPMX][IGPSRIHXLMWFMRHMRKWMXIMRXSXLI´986SJEPYGMJIVEWI
VITSVXIVKIRIHVMZIRF]EGSRWXMXYXMZITVSQSXIVT7(* ERHEREP]^IHPYGMJIVEWI
I\TVIWWMSRERHSJEGSRXVSPTPEWQMHMRXLILYQERIRHSXLIPMEPGIPPPMRI)'6*
-RXLIWIQM6I\TVIWWMRKGIPPWPYGMJIVEWII\TVIWWMSRHVMZIRJVSQEVITSVX
IV GSRWXVYGX PEGOMRK E QM62% WIIH WIUYIRGI T1-6 [EW RSX EJJIGXIH [LIR
QM6 [EW WMPIRGIH YWMRK ER ERXEKSQMV ETTVSEGL ERXE -R GSRXVEWX E
VITSVXIVKIRIGEVV]MRKEWMRKPITIVJIGXQM6FMRHMRKWMXIT1323 [EWJYPP]
EGXMZI[LIRQM6[EWWMPIRGIHFYXMXWI\TVIWWMSR[EWWXVSRKP]VIHYGIHMRXLI
TVIWIRGISJEGSRXVSPWGVEQFPIQMVWGV 8LIWIWXYHMIWGSR½VQXLIJYRGXMSREPMX]
SJ QM6 MR )'6* GIPPW 0MOI[MWI XLI TVIWIRGI SJ XLI TYXEXMZI 7(*
QM6 FMRHMRK WMXI MR XLI VITSVXIV GSRWXVYGX PIH XS E VIHYGXMSR SJ
PYGMJIVEWII\TVIWWMSR MR QM6 I\TVIWWMRK GIPPW *MKYVI % WGVEQFPI ZIVWYW
ERXEKSQMV4 %WMRKPIQMWQEXGLMRXLIWIIHWIUYIRGISJXLITYXEXMZI
7(*QM6FMRHMRKWMXI'XS+EXTSWMXMSRSJXLIWIIHWIUYIRGIT7(
*QQ EPPIZMEXIH QM6HITIRHIRX VITVIWWMSR SJ VITSVXIV KIRI I\TVIWWMSR
GSR½VQMRKXLITVIWIRGIERHWTIGM½GMX]SJXLIQM6FMRHMRKWMXIMRXLI´986
SJXLI7(*Q62%8SZEPMHEXIXLIVIKYPEXSV]IJJIGXSJQM6SRIRHSKIRSYW
7(*TVSXIMRI\TVIWWMSRMRIRHSXLIPMEPGIPPWLYQERYQFMPMGEPZIMRIRHSXLIPMEP
GIPPW ,9:)' [IVI XVIEXIH [MXL ERXEKSQMV SV WGVEQFPIQMV EJXIV [LMGL
7(*TVSXIMRI\TVIWWMSR[EWHIXIVQMRIHF];IWXIVRFPSXEREP]WMW%QEVOIH
MRGVIEWISJ7(*TVSXIMRI\TVIWWMSR[EWSFWIVZIHMR,9:)'XVIEXIH[MXLER
XEKSQMVEWGSQTEVIHXSWGVEQFPIQMV*MKYVI&
2I\X[IEWWIWWIHXLITSXIRXMEPSJQM6XSVIKYPEXIXLII\XIRXF][LMGL
IRHSXLIPMEPGIPPHIVMZIH7(*EXXVEGXWTVSKIRMXSVGIPPW8SXLMWIRHGSRHMXMSRIH
QIHMYQ HIVMZIH JVSQ ,9:)' MRGYFEXIH [MXL ERXEKSQMV SV WGVEQFPIQMV
[EWETTPMIHXSXLIPS[IVGSQTEVXQIRXSJEXVERW[IPPW]WXIQERHYQFMPMGEPGSVH
FPSSHHIVMZIH'(GIPPW[IVITPEGIHMRXLIYTTIVGSQTEVXQIRX%WWLS[R
MR *MKYVI ' WMPIRGMRK SJ QM6 MR ,9:)'IRLERGIH QMKVEXMSR SJ '( GIPPWGSQTEVIHXSXLIWGVEQFPIQMVGSRXVSP4 1SVISZIVRIYXVEPM^EXMSR
SJ '<'6 I\TVIWWIH F] XLI '( GIPPW [MXL E JYRGXMSRFPSGOMRK ERXMFSH]
Ơ'<'6 ÜKQ0 HIGVIEWIH GIPP QMKVEXMSR XS[EVHW XLI GSRHMXMSRIH WYTIVREXERXHIVMZIHJVSQERXEKSQMVXVIEXIH,9:)'FEGOXSXLIWGVEQFPI
GSRXVSPPIZIPW4 %WEGSRXVSPQE\MQEPGIPPYPEVQMKVEXMSR[EWSFWIVZIH
YWMRKQIHMYQGSRXEMRMRKVIGSQFMRERXLYQER7(*RKQ0 [LMGL[EWWMK
RM½GERXP]VIHYGIHF]Ơ'<'6*MKYVI'4 8LIWIHEXEWYKKIWXXLEXWYT
TVIWWMSRSJQM6MRIRHSXLIPMEPGIPPWMRZMXVSGEREYKQIRXI\TVIWWMSRSJJYRG
XMSREP7(*PIEHMRKXSIPIZEXIHTVSKIRMXSVGIPPQMKVEXMSR
1M6EJJIGXWTVSKIRMXSVGIPPQSFMPM^EXMSRZME7(*MRZMZS )RHSXLIPMEPQM6I\TVIWWMSRERHJYRGXMSREPMX]MWGSRWIVZIHMRQYVMRI)'
FSXLMRZMZS?AERHMRZMXVSWYTTPIQIRXEV]*MKYVIW7ERH7 8SMRZIWXMKEXI
XLIVIKYPEXSV]VSPISJQM6SR7(*HITIRHIRXTVSKIRMXSVGIPPQSFMPM^EXMSR
MR ZMZS [I ETTPMIH WMRKPI XEMP ZIMR MRNIGXMSRW SJ ERXEKSQMV SV XLI GSRXVSP
WGVEQFPIQMV MRXS [MPH X]TI ;8 QEPI '&P QMGI R! 8IR HE]W EJXIV
MRNIGXMSR[IIRYQIVEXIHXLIRYQFIVSJGMVGYPEXMRK7GETSWMXMZIERHPMRIEKI
RIKEXMZI 7GE0MR GIPPW F] *%'7 EREP]WMW 7YVTVMWMRKP] [LIR [I I\TVIWWIH
XLIGMVGYPEXMRK7GE0MRGIPPRYQFIVWEWETIVGIRXEKISJ0MRPIYOSG]XIW[I
SFWIVZIHEWMKRM½GERXHIGVIEWIMRGMVGYPEXMRK7GE0MRGIPPWMRXLIQMGIMR[LMGL
QM6 LEH FIIR WMPIRGIH F] ERXEKSQMV EHQMRMWXVEXMSR EW GSQTEVIH XS
XLIWGVEQFPIQMVXVIEXIHGSRXVSPW*MKYVI%4 8LMWSFWIVZEXMSRGSYPHFI
I\TPEMRIHF]XLIJEGXXLEXERXEKSQMVXVIEXIHQMGIEPWSHMWTPE]IHEWMKRM½GERX
MRGVIEWI MR XSXEP [LMXI FPSSH GIPP ;&' GSYRX *MKYVI & 4 7YGL ER
MRGVIEWIMWGSRWMWXIRX[MXLVITSVXWWLS[MRKXLEXMRGVIEWIH7(*PIZIPWMRXLI
GMVGYPEXMSRVIWYPXMRERIPIZEXMSRSJ;&'W?A%WWYGL[LIR[II\TVIWWIHXLI
RYQFIV SJ GMVGYPEXMRK 7GE0MR GIPPW TIV Q0 FPSSH XLI HMJJIVIRGIW FIX[IIR
ERXEKSQMVERHWGVEQFPIQMVXVIEXIHERMQEPW[EWPSWXMRHMGEXMRKXLEXXLIVIMW
RSW]WXIQMGVSPIJSVQM6MR7GE0MRTVSKIRMXSVGIPPQSFMPM^EXMSRMRZMZS
*MKYVI'
7MRGIMWGLIQMGIRHSXLIPMEPGIPPWLEZIFIIRIWXEFPMWLIHXSYTVIKYPEXI7(*?A
*MKYVI QM6 EJJIGXW XLI I\TVIWWMSR ERH JYRGXMSR SJ 7(*
MRZMXVS
% )'6* [IVI TVIMRGYFEXIH [MXL WGVEQFPIQMV SV ERXEKSQMV ERH XVERWJIGXIH [MXL T1-6 EW RIKEXMZI GSRXVSP T1323 EW TSWMXMZI GSRXVSP T7(*
[MXL XLI WIUYIRGI SJ XLI 7(* QM6 FMRHMRK WMXI SV E ZEVMERX [MXL E WMRKPI
QMWQEXGLMRXLIWIIHWIUYIRGIT7(*QQ )\TIVMQIRX[IVITIVJSVQIHMRXVMTPMGEXI
ERHHEXEEVII\TVIWWIHEWQIERZEPYIWWXERHEVHIVVSVSJXLIQIER& ,9:)'
[IVIMRGYFEXIHJSVLSYVW[MXLERXEKSQMVSVWGVEQFPIQMVERHGSRHMXMSRIH
GIPPWYTIVREXERXWWYFNIGXIHXS;IWXIVRFPSXEREP]WMW5YERXMXEXMZIEREP]WMWSJ7(*
FERHMRXIRWMX]SR;IWXIVRFPSX[IVITIVJSVQIHYWMRK-QEKI.ERHRSVQEPM^IHJSV
ơEGXMR[MXLXLIERXEKSQMVFERHWIXEX ' ,9:)'R! [IVIXVIEXIH
[MXLERXEKSQMVWGVEQFPIQMVSVZILMGPIERHXLIGSRHMXMSRIHQIHME[IVIYWIH
XS WXYH] XLI GLIQSXEGXMG EGXMZMX] XS[EVHW LYQER LIQEXSTSMIXMG '( GIPPW MR
ZMXVS6IGSQFMRERX7(*[EWYWIHEWTSWMXMZIGSRXVSPERHXLIGSRXVMFYXMSRSJXLI
7(*VIGITXSV'<'6 [EWWXYHMIH[MXLXLIYWISJEJYRGXMSRFPSGOMRKERXMFSH]
Ơ'<'6 R!
% ns P<0.01 P<0.01 ns
0 50 100
anta-126 scr
relative luciferase expression (%)
pMIR pMONO pSDF pSDFmm
'
0 10 20 30
migration compared to control (%)
P<0.05 P<0.001
P<0.01
addition SDF-1 vehicle scram anta- 126
DCXCR4 - + - + - + - +
& anta-126 ȕ ȕ-actin SDF-1 S scr
100 12 SDF-1/ S ȕ-actin (%)
ERHWYTTPIQIRXEV]*MKYVI7 ;IMRZIWXMKEXIH[LIXLIVERMWGLIQMGWXMQYPYW
MRGSQFMREXMSR[MXLEXXIRYEXMSRSJQM6PIZIPW[SYPHQSHYPEXITVSKIRMXSV
GIPP PIZIPW MR QMGI8LIVIJSVI [I MRNIGXIH QEPI;8 QMGI R! [MXL IMXLIV
ERXEKSQMVSVWGVEQFPIQMVERHXLIRI\XHE]XLMWTVSGIHYVI[EWJSPPS[IH
F]XLIMRHYGXMSRSJYRMPEXIVEPLMRHPMQFMWGLIQME,0- 8IRHE]WEJXIVMRHYGXMSR
SJ,0-XLIERMQEPW[IVIWEGVM½GIHERHWIVYQPIZIPWSJ7(*[IVIEWWIWWIHF]
)0-7%%PXLSYKLW]WXIQMG7(*PIZIPW[IVIIPIZEXIHMRERXEKSQMVXVIEXIH
QMGIEWGSQTEVIHXSGSRXVSPWRSWMKRM½GERGI[EWVIEGLIH*MKYVI%4!
2IZIVXLIPIWW[LIR[II\EQMRIHXLIMRZMZSQSFMPM^EXMSRSJ7GE0MRGIPPWXS
XLIGMVGYPEXMSREJXIVMWGLIQME[ISFWIVZIHERJSPHMRGVIEWISJ7GE0MRGIPPW
I\TVIWWIHEWTIVGIRXEKISJXSXEP0MRPIYOSG]XIWMRXLIFPSSHSJERXEKSQMV
XVIEXIH ERMQEPW EW GSQTEVIH XS WGVEQFPIQMVXVIEXIH GSRXVSP ERMQEPW *MKYVI
&4 8LIEFWSPYXIRYQFIVSJ7GE0MRGIPPWTIVQ0FPSSH[EWVEMWIH
JVSQMRXLIGSRXVSPKVSYTXSMRXLIERXEKSQMVXVIEXIH
ERMQEPW%WXLISFWIVZIHIJJIGXWWYKKIWXIHERMWGLIQMEHITIRHIRXIPIZEXMSRSJ
7(*[IRI\XMRZIWXMKEXIH[LIXLIVXLIMWGLIQMGKEWXVSGRIQMYWQYWGPIGSYPH
FIETSXIRXMEPGSRXVMFYXSVXSXLIIPIZEXIHPIZIPWSJGMVGYPEXMRK7(*8LIVIJSVI
[ITIVJSVQIHFSXLHIXEMPIHUYEPMXEXMZIERHUYERXMXEXMZIMQQYRSLMWXSGLIQMGEP
EREP]WMW SJ 7(* I\TVIWWMSR MR VIPEXMSR XS '(TSWMXMZI GETMPPEVMIW
-RHIIH[ISFWIVZIHEGPIEVGSPSGEPM^EXMSRSJ7(*ERH'(I\TVIWWMSRMR
WIGXMSRWSJXLIMWGLIQMGQYWGPI*MKYVI' *YVXLIVQSVIXLIMWGLIQMGQYWGPISJ
ERXEKSQMVXVIEXIHERMQEPWWLS[IHELMKLIVTIVGIRXEKISJHSYFPITSWMXMZI
GETMPPEVMIW *MKYVI ( 4 EW STTSWIH XS WGVEQFPIQMVXVIEXIH GSRXVSPW
8LIWIHEXEMQTP]XLEXXLIQMPHP]IPIZEXIHPIZIPWSJ7(*TVSXIMRMRXLIGMVGYPEXMSR
*MKYVIQM6HSIWRSXWSPIP]EJJIGXQSFMPM^EXMSRSJ7GE0MR GIPPWMRZMZS
% 8IR HE]W EJXIV MRNIGXMSR SJ WGVEQFPIQMV SV ERXEKSQMV 7GE0MR GIPPW MR
[LSPI FPSSH [IVI EREP]^IH F] *%'7 I\TVIWWIH EW TIVGIRXEKI SJ XSXEP 0MR PIYOSG]XIW & 8IR HE]W EJXIV MRNIGXMSR SJ WGVEQFPIQMV SV ERXEKSQMV XSXEP
[LMXIFPSSHGIPPGSYRXWTIVQ0[EWHIXIVQMRIH' 8IRHE]WEJXIVWGVEQFPIQMVSV ERXEKSQMVMRNIGXMSR 7GE0MR GIPPW [IVI EREP]^IH F] *%'7 MR [LSPI FPSSH
I\TVIWWIHEWXSXEPRYQFIVMRQ0FPSSH
% %
scr anta-126 0
1 2 3 4 5
Sca+/Lin- cells per Lin+ leukocytes (%)
P<0.05
scr anta-126 0
5 10 15
white blood cells (106/mL) P<0.01
scr anta-126 0.0
0.1 0.2
Sca+/Lin- cell per mL blood
& '
YTSR ERXEKSQMVXVIEXQIRX QE] FI E VI¾IGXMSR SJ XLI PSGEPP] IRLERGIH
I\TVIWWMSRSJ7(*TVSXIMRMRXLIZIWWIPWSJMWGLIQMGXMWWYI
(MWGYWWMSR
%JYRGXMSRJSVQM6MRERKMSKIRMGTVSGIWWIWMRZEWGYPEVQEMRXIRERGIERH
HYVMRKHIZIPSTQIRXLEWFIIRWLS[RMRERYQFIVSJTYFPMGEXMSRWHIQSRWXVEXMRK
XLEX PSWW SJ QM6 IMXLIV MR ORSGOSYX QSHIPW SV QIHMEXIH F] XVIEXQIRX
[MXLERXEKSQMVWPIEHWXSWXVYGXYVEPMQTEMVQIRXSJXLIZEWGYPEVFIH?A-R
XLIGYVVIRXWXYH][ITVSZMHIIZMHIRGIJSVEZEWGYPSKIRMGVSPIJSVQM6MR
VIKYPEXMRK XLI QSFMPM^EXMSR SJ IRHSXLIPMEP TVSKIRMXSV GIPPW ZME XLI VIPIEWI SJ
GLIQSOMRI 7(* JVSQ MWGLIQMG IRHSXLIPMEP GIPPW -R ZMXVS XLI MRGVIEWIH WIGVIXMSR SJ 7(* YTSR WMPIRGMRK SJ QM6 [EW WYJ½GMIRX XS WXMQYPEXI XLI
QMKVEXMSRSJLYQER'(WXIQGIPPW-RQMGILS[IZIVW]WXIQMGWMPIRGMRK[MXL
EWMRKPIXEMPZIMRMRNIGXMSRSJERXEKSQMV[EWRSXWYJ½GMIRXXSVEMWIXLIPIZIPW
SJGMVGYPEXMRKQYVMRI7GE0MRTVSKIRMXSVGIPPW,S[IZIVMRGSQFMREXMSR[MXL
XLI PMKEXMSR SJ XLI JIQSVEP EVXIV] [I HIQSRWXVEXIH ER MRGVIEWI MR GMVGYPEXMRK
7GE0MR GIPPW JSPPS[MRK QM6 WMPIRGMRK WXVSRKP] WYKKIWXMRK XLEX XMWWYI
MWGLIQMEMWRIIHIHXSVIZIEPXLIVIKYPEXSV]VSPISJQM6MRZMZS
3YVHEXEWYKKIWXXLEXIPIZEXIHRYQFIVWSJGMVGYPEXMRK7GE0MRGIPPWMRXLI
ERXEKSQMVXVIEXIH ERMQEPW EVI XLI VIWYPX SJ 7(* QIHMEXIH QSFMPM^EXMSR SJ
XLIWIGIPPWJSPPS[MRKMWGLIQME8LMWMWWYTTSVXIHF]XLIJEGXXLEX7(*TVSXIMR
I\TVIWWMSRMWEPWSYTVIKYPEXIHMRXLIIRHSXLIPMEPGIPPWSJXLIMWGLIQMGXMWWYIEW
*MKYVI %& QM6 GER EPXIV 7(*I\TVIWWMSR ERH MRHYGI
TVSKIRMXSVQSFMPM^EXMSREJXIVMWGLIQMGMRNYV]MRZMZS
% 8IR HE]W EJXIV ,0- ERXEKSQMVXVIEXIH QMGI WLS[IH RSRWMKRM½GERXP]
IPIZEXIH PIZIPW SJ 7(* MR XLI WIVYQ EW GSQTEVIH XS WGVEQFPIQMVXVIEXIH
GSRXVSPW R! & 8IR HE]W EJXIV ,0- ERH MRNIGXMSR SJ WGVEQFPIQMV SV ERXEKSQMV 7GE0MR GIPPW [IVI QIEWYVIH MR [LSPI FPSSH I\TVIWWIH EW TIVGIRXEKISJXSXEP0MRPIYOSG]XIW
% &
P=0.19
scr
scr anta-126anta 126 0.0
0.5 1.0 1.5 2.0
SDF-1 (ng/mL)
0.0 0.5 1.0 1.5
Sca-1+ /Lin- cells per Lin+ leukocytes (%) P<0.005
B
scr
scr anta-126anta 126
[IPP EW MR XLI TIVMTLIVEP GMVGYPEXMSR 2IZIVXLIPIWW HI½RMXMZI TVSSJ SJ E GEYWEP
VSPI SJ QM6 MR 7(* HITIRHIRX QSFMPM^EXMSR SJ TVSKIRMXSV GIPPW [SYPH
VIUYMVIERMRZMZSFPSGOEHIMRXLI7(*'<'6E\MW*SVXLMWMRLMFMXSVWSJXLI
MRXIVEGXMSRSJ7(*[MXL'<'6WYGLEW%1(GSYPHFIEHQMRMWXVEXIH
XS ERXEKSQMV XVIEXIH QMGI ERH YWIH XS EPPIZMEXI XLI IPIZEXIH TVSKIRMXSV
GIPP QSFMPM^EXMSRJSPPS[MRKMWGLIQME8LMWETTVSEGLMWLS[IZIVGSQTPMGEXIHF]
XLIHMVIGXIJJIGXWSJXLIWIMRLMFMXSVWSRXLIIKVIWWSJTVSKIRMXSVGIPPWJVSQXLI
FSRIQEVVS[[LMGLGSYPHTSXIRXMEPP]SZIVVMHIXLI7(*IJJIGXWIPMGMXIHF]XLI
IRHSXLIPMYQ MR XLI MWGLIQMG XMWWYIW MR XLI TIVMTLIV] ?A %PWS MQTEMVIH
TVSKIRMXSVLSQMRKXSXLIMWGLIQMGXMWWYISVXLIWTPIIRGERRSXFIJYPP]I\GPYHIH
?A
%R MRXIVEGXMSR FIX[IIR QM6 ERH 7(* LEW TVIZMSYWP] FIIR WLS[R XS
MRGVIEWI QM6 YTXEOI SJ IRHSXLIPMEP GIPPHIVMZIH ETSTXSXMG FSHMIW F]
IRHSXLIPMEPGIPPW8LMWVIWYPXIHMRMRGVIEWIH7(*I\TVIWWMSRXLVSYKLMRLMFMXMSR
SJ 6IKYPEXSV SJ +TVSXIMR WMKREPMRK 6+7 ?A -R GSRXVEWX SYV WXYHMIW
MQTPMGEXIXLEXXLIEFVSKEXMSRSJQM6MWEWWSGMEXIH[MXLMRGVIEWIHI\TVIWWMSR
SJ7(*WYKKIWXMRKXLEXQM62%WGSYPHWIVZIEWEFMSPSKMGEPVLISWXEX[MXLXLI
VIWTSRWIQEKRMXYHISJFMSPSKMGEPTEXL[E]WFIMRKHITIRHIRXSRXLIGSRXI\XERH
WSYVGISJXLII\XIVREPWXMQYPYW
6IGIRXP] MX LEW FIIR HIQSRWXVEXIH XLEX QM62%W EVI TVIWIRX MR XLI
GMVGYPEXMSRERHXLEXEPXIVEXMSRWMRXLITVS½PISJTPEWQESVWIVYQQM62%WGER
FIEWWSGMEXIH[MXLHMWIEWIWXEXIW?A8LIWIIEVP]VITSVXWQEMRP]HMWTPE]IHE
PMROFIX[IIRGMVGYPEXMRKQM62%WERHGERGIV[LMPIWYFWIUYIRXWXYHMIWLEZIEPWS
VIZIEPIHEGPIEVEWWSGMEXMSRSJGMVGYPEXMRKQM62%W[MXLGEVHMSZEWGYPEVHMWIEWI
?A7MRGIIRHSXLIPMEPMRNYV]MWGSRWMHIVIHSRISJXLILEPPQEVOWSJTEXMIRXW
EXVMWOJSVGEVHMSZEWGYPEVHMWIEWIERHYTSRMRNYV]IRHSXLIPMEPGIPPWGERWIGVIXI
QM62%GSRXEMRMRKQMGVSZIWMGPIW?AMXMWSJMRXIVIWXXSEHHVIWWXLIZEPYI
SJIRHSXLIPMEPGIPPHIVMZIHGMVGYPEXMRKQM62%WEWFMSQEVOIVWJSVGEVHMSZEWGYPEV
HMWIEWI -RHIIH X[S VIGIRX GPMRMGEP WXYHMIW VIZIEPIH E HIGVIEWI MR GMVGYPEXMRK
QM6PIZIPWMRTEXMIRXW[MXLGSVSREV]EVXIV]HMWIEWI'%( ?AERHHMEFIXIW
QIPPMXYW(1 ?A-X[EWWYKKIWXIHXLEXPS[IVIHPIZIPWSJQM6MR(1
TEXMIRXW QMKLX FI I\TPEMRIH F] XLI JEGX XLEX LMKL KPYGSWI PIZIPW GER PIEH XS E
HIGVIEWISJXLIQM6GSRXIRXMRXLIIRHSXLIPMEPTEVXMGPIW[LMPIGIPPYPEVQM62%
PIZIPWVIQEMRIHYREPXIVIH?A3RIQE]WTIGYPEXIXLEXPS[IVMRKSJGMVGYPEXMRK
QM6PIZIPWQMKLXFIEWTIGM½GWMKREPSJXLIMRNYVIHIRHSXLIPMYQXSMRGVIEWI
XLII\TVIWWMSRPIZIPWSJHMWXMRGXQM6XEVKIXWXLEXEVIGVMXMGEPJSVXLIMRXIKVMX]
SJXLIIRHSXLIPMYQ
;I WYKKIWX LIVI XLEX QM6 JYRGXMSRW EW E VIKYPEXSV SJ IRHSXLIPMEP
LSQISWXEWMW *MKYVI -R XLI LIEPXL] ZMEFPI IRHSXLIPMYQ QM6 MW VIEHMP]
I\TVIWWIH ERH WIVZIW XS HS[R VIKYPEXI 746)( ERH 4-/6 FSXL SJ [LMGL
EVI MRLMFMXSVW SJ ERKMSKIRMG ERH GIPP WYVZMZEP WMKREPW MR VIWTSRWI XS ZEWGYPEV
IRHSXLIPMEP GIPP KVS[XL JEGXSV :)+* ? A 8LMW GSRHMXMSR JEZSVW XLI
*MKYVI '( QM6 GER EPXIV 7(*I\TVIWWMSR ERH MRHYGI TVS
KIRMXSVQSFMPM^EXMSREJXIVMWGLIQMGMRNYV]MRZMZS
' -QQYRSLMWXSGLIQMGEPQMGVSKVETLWSJMQQYRSLMWXSGLIQMWXV]SJXLIKEWXVSGRIQMYW QYWGPIEJXIV,0-WLS[IHGSPSGEPM^EXMSRSJ7(*ERH'(7(*[EWZMWYEPM^IH
[MXLEPI\EKVIIR ERH'([MXLEPI\EVIH 2YGPIM[IVIWXEMRIH[MXL
(%4- ERH WLS[R MR FPYI 2SQEVWO] GSRXVEWX MQEKIW WLS[ QYWGPI XMWWYI 7GEPI
FEVW VITVIWIRX ÜQ ( 5YERXM½GEXMSR SJ QMGVSWGSTMG MQEKIW HMWTPE]IH XLEX
ERXEKSQMVXVIEXIH QMGI LEZI MRGVIEWIH PIZIPW SJ 7(* TSWMXMZI GETMPPEVMIW EW
TIVGIRXEKISJXSXEPRYQFIVSJGETMPPEVMIWR! '
( P<0.01
scr
scr anta-126anta 126 0
20 40 60 80
SDF-1 positive capillaries (%)
ERKMSKIRMGVIWTSRWIXSMRNYV]EW7(*I\TVIWWMSRMWGSRGSQMXERXP]VITVIWWIH
F]QM63RXLISXLIVLERHYRHIVGSRHMXMSRWEWWSGMEXIH[MXLIRHSXLIPMEP
GIPP H]WJYRGXMSR SV WIRIWGIRGI E HIGVIEWI MR QM6 PIZIPW [SYPH MRLMFMX
ERKMSKIRMG ERH GIPP WYVZMZEP WMKREPW *YVXLIVQSVI XLMW GSRHMXMSR JEZSVW XLI
I\TVIWWMSR SJ 7(* ERH:'%1 ?A SJ XLI EJJIGXIH IRHSXLIPMEP GIPPW ERH
WYTTSVX VIIRHSXLIPMEPM^EXMSR F] E ZEWGYPSKIRMG VIWTSRWI XLVSYKL XLI
VIGVYMXQIRX ERH WYFWIUYIRX EHLIWMSR SJ ZEWGYPEV TVSKIRMXSV GIPPW %W FSRI
QEVVS[HIVMZIH'(GIPPW[IVIVIGIRXP]HIQSRWXVEXIHXSVITVIWIRXEQSVI
JYRGXMSREP)4'TSTYPEXMSRXLER7GE0MRTVSKIRMXSVGIPPWJYXYVIWXYHMIWQE]
MRGPYHIXLIWITVSKIRMXSVGIPPTSTYPEXMSREW[IPP?A
%PXLSYKL WTIGYPEXMZI SYV QSHIP MW WYTTSVXIH F] XLI SFWIVZIH HIGVIEWIH
PIZIPWSJGMVGYPEXMRKQM6MR(1ERH'%(TEXMIRXW?AERHIPIZEXIH
PIZIPWSJ7(*MRTEXMIRXW[MXLEGYXIGSVSREV]W]RHVSQI%'7 ?A*YXYVI
WXYHMIWMRTEXMIRXGSLSVXW[MPPTVSZMHIMRWMKLXEWXS[LIXLIVXLIHS[RVIKYPEXMSR
SJGMVGYPEXMRKQM6GERFIGSVVIPEXIH[MXLMRGVIEWIHPIZIPWSJ7(*
-R GSRGPYWMSR FSXL MR ZMXVS ERH MR ZMZS QM6 GER VIKYPEXI XLI I\TVIWWMSR
SJ7(*MRIRHSXLIPMEPGIPPW[LMGLQE]PIEHXSXLIQSFMPM^EXMSRSJ7GE0MR TVSKIRMXSV GIPPW MRXS XLI TIVMTLIVEP GMVGYPEXMSR JSPPS[MRK GSRHMXMSRW SJ EGYXI
MWGLIQME%W XLI IRHSXLIPMEP QM6 VIKYPEXIW FSXL JIEXYVIW SJ ERKMSKIRIWMW
EW [IPP EW ZEWGYPSKIRIWMW E VIKYPEXSV] VSPI JSV XLMW QM62% MR IRHSXLIPMEP
LSQISWXEWMWMWTVSTSWIH
*MKYVIQM6EGXWEWEZEWGYPSKIRMGW[MXGL
-R ZMEFPI IRHSXLIPMYQ QM6 MRLMFMXW XLI I\TVIWWMSR SJ 746)( ERH 4-/6
XLIVIF]JEGMPMXEXMRK:)+*HITIRHIRXERKMSKIRIWMW*YVXLIVQSVIXLII\TVIWWMSRSJ
7(*ERH:'%1EVIMRLMFMXIH;LIRQM6MWPSWX746)(ERH4-/6EVI
YTVIKYPEXIHXLIVIF]FPSGOMRKERKMSKIRIWMWEXXLIWEQIXMQI7(*ERH:'%1
PIZIPW EVI YT VIKYPEXIH ERH XLMW IPIZEXMSR WYFWIUYIRXP] PIEHW XS ER MRGVIEWIH
QSFMPM^EXMSRERHEHLIWMSRSJFSRIQEVVS[HIVMZIHTVSKIRMXSVW
progenitor cell mobilization and adhesion viable endothelium
SPRED-1 PI3KR2
VEGFR
angiogenesis survival
miR-126
SDF-1
dysfunctional endothelium
SPRED-1 PI3KR2 VEGFR
miR-126
SDF-1 VCAM-1 VCAM-1
7SYVGIWSJJYRHMRK
'Z7 MW WYTTSVXIH F] XLI 2IXLIVPERHW ,IEVX *SYRHEXMSR KVERX RYQFIV
& 11ERH6&EVIWYTTSVXIHF]XLI(YXGL/MHRI]*SYRHEXMSRKVERX
RYQFIV ' ERH ' VIWTIGXMZIP] )ZH: MW WYTTSVXIH F] XLI
8VERWPEXMSREP)\GIPPIRGIMR6IKIRIVEXMZI1IHMGMRI8I61 7QEVX1M\4VSKVEQSJ
XLI2IXLIVPERHW1MRMWXV]SJ)GSRSQMG%JJEMVWERHXLI2IXLIVPERHW1MRMWXV]SJ
)HYGEXMSR'YPXYVIERH7GMIRGI%.Z>MWWYTTSVXIHF]EKVERXJVSQXLI+IR^]QI
6IREP-RRSZEXMSRW4VSKVEQ
6IJIVIRGIW
6EFIPMRO8.HI&SIV,'ZER>SRRIZIPH%.)RHSXLIPMEPEGXMZEXMSRERHGMVGYPEXMRKQEVOIVWSJ
IRHSXLIPMEPEGXMZEXMSRMROMHRI]HMWIEWI2EX6IZ2ITLVSP
7GL[EVX^71&IRHMXX)4'PYWXIVMRKSJVITPMGEXMRKGIPPWMRESVXMGIRHSXLIPMYQ4VSG2EXP%GEH
7GM97%
/YV^(.(IGEV]7,SRK=8VMZMIV)%OLQIHSZ%)VYWEPMQWO].('LVSRMGS\MHEXMZIWXVIWW
GSQTVSQMWIWXIPSQIVIMRXIKVMX]ERHEGGIPIVEXIWXLISRWIXSJWIRIWGIRGIMRLYQERIRHSXLI
PMEPGIPPW.'IPP7GM
3THIR&YMNW.1YWXIVW1:IVVMTW84SWX.%&VEEQ&ZER6MIP21EXLIQEXMGEPQSHIPMRKSJ
ZEWGYPEV IRHSXLIPMEP PE]IV QEMRXIRERGI XLI VSPI SJ IRHSXLIPMEP GIPP HMZMWMSR TVSKIRMXSV GIPP
LSQMRKERHXIPSQIVIWLSVXIRMRK%Q.4L]WMSP,IEVX'MVG4L]WMSP,,
*SXIMRSW+,Y=<MES51IX^PIV&<Y56ETMHIRHSXLIPMEPXYVRSZIVMREXLIVSWGPIVSWMW
TVSRIEVIEWGSMRGMHIW[MXLWXIQGIPPVITEMVMRETSPMTSTVSXIMR)HI½GMIRXQMGI'MVGYPEXMSR
%WELEVE88EOELEWLM8 1EWYHE , /EPOE ' 'LIR ( -[EKYVS , IX EP:)+* GSRXVMFYXIW XS
TSWXREXEPRISZEWGYPEVM^EXMSRF]QSFMPM^MRKFSRIQEVVS[HIVMZIHIRHSXLIPMEPTVSKIRMXSVGIPPW
)1&3.
=EQEKYGLM . /YWERS /* 1EWYS 3 /E[EQSXS% 7MPZIV 1 1YVEWE[E 7 IX EP 7XVSQEP GIPP
HIVMZIH JEGXSV IJJIGXW SR I\ ZMZS I\TERHIH IRHSXLIPMEP TVSKIRMXSV GIPP VIGVYMXQIRX JSV
MWGLIQMGRISZEWGYPEVM^EXMSR'MVGYPEXMSR
1EWWFIVK7/SRVEH-7GLYV^MRKIV/0SVIR^17GLRIMHIV7>SLPRLSIJIV(IXEP4PEXIPIXW
WIGVIXIWXVSQEPGIPPHIVMZIHJEGXSVEPTLEERHVIGVYMXFSRIQEVVS[HIVMZIHTVSKIRMXSVGIPPW
XSEVXIVMEPXLVSQFMMRZMZS.)\T1IH
,EXXSVM/,IMWWMK&6E½M78LIVIKYPEXMSRSJLIQEXSTSMIXMGWXIQGIPPERHTVSKIRMXSVQSFMPM
^EXMSRF]GLIQSOMRI7(*0IYO0]QTLSQE
HI&SIV,',SZIRW11ZER3IZIVIR6MIXHMNO%17RSIT.(HI/SRMRK).8EQWQE.8IXEP
,YQER'(/(6GIPPWEVIKIRIVEXIHJVSQGMVGYPEXMRK'(GIPPWEJXIVMQQSFMPM^EXMSR
SREGXMZEXIHTPEXIPIXW%VXIVMSWGPIV8LVSQF:EWG&MSP
,MEWE / -WLMFEWLM 1 3LXERM / -RSYI 7 >LES 5 /MXEQSXS 7 IX EP +IRI XVERWJIV SJ WXVS
QEPGIPPHIVMZIHJEGXSVEPTLEIRLERGIWMWGLIQMGZEWGYPSKIRIWMWERHERKMSKIRIWMWZMEZEWGYPEV
IRHSXLIPMEPKVS[XLJEGXSVIRHSXLIPMEPRMXVMGS\MHIW]RXLEWIVIPEXIHTEXL[E]RI\XKIRIVEXMSR
GLIQSOMRIXLIVET]JSVXLIVETIYXMGRISZEWGYPEVM^EXMSR'MVGYPEXMSR
%WOEVM%89R^IO74STSZMG>&+SPHQER'/*SVYHM*/MIHVS[WOM1IXEP)JJIGXSJWXVSQEP
GIPPHIVMZIHJEGXSVSRWXIQGIPPLSQMRKERHXMWWYIVIKIRIVEXMSRMRMWGLEIQMGGEVHMSQ]STE
XL]0ERGIX
8ITTIV31'ETPE.1+EPMERS6('IVEHMRM(.'EPPEKLER1./PIMRQER1)IXEP%HYPXZEWGY
PSKIRIWMWSGGYVWXLVSYKLMRWMXYVIGVYMXQIRXTVSPMJIVEXMSRERHXYFYPM^EXMSRSJGMVGYPEXMRKFSRI
QEVVS[HIVMZIHGIPPW&PSSH
/PSSWXIVQER;44PEWXIVO6,8LIHMZIVWIJYRGXMSRWSJQMGVS62%WMRERMQEPHIZIPSTQIRX
ERHHMWIEWI(IZ'IPP
<MI<0Y./YPFSOEW).+SPYF861SSXLE:0MRHFPEH8SL/IXEP7]WXIQEXMGHMWGSZIV]SJ
VIKYPEXSV]QSXMJWMRLYQERTVSQSXIVWERH´986WF]GSQTEVMWSRSJWIZIVEPQEQQEPW2E
XYVI
9VFMGL'/YILFEGLIV%(MQQIPIV76SPISJQMGVS62%WMRZEWGYPEVHMWIEWIWMR¾EQQEXMSR
ERHERKMSKIRIWMW'EVHMSZEWG6IW
/YILFEGLIV%9VFMGL'>IMLIV%1(MQQIPIV76SPISJ(MGIVERH(VSWLEJSVIRHSXLIPMEP
QMGVS62%I\TVIWWMSRERHERKMSKIRIWMW'MVG6IW
*EWEREVS4(´%PIWWERHVE=(M7XIJERS:1IPGLMSRRE66SQERM74SQTMPMS+IXEP1MGVS
62%QSHYPEXIWIRHSXLIPMEPGIPPVIWTSRWIXSL]TS\MEERHMRLMFMXWXLIVIGITXSVX]VSWMRI
OMREWIPMKERH)TLVMR%.&MSP'LIQ
'LIR=+SVWOM(,6IKYPEXMSRSJERKMSKIRIWMWXLVSYKLEQMGVS62%QM6E XLEXHS[R
VIKYPEXIWERXMERKMSKIRMGLSQISFS\KIRIW+%<ERH,3<%&PSSH
7YEVI^=*IVRERHI^,IVRERHS'4SFIV.77IWWE;'(MGIVHITIRHIRXQMGVS62%WVIKYPEXI
KIRII\TVIWWMSRERHJYRGXMSRWMRLYQERIRHSXLIPMEPGIPPW'MVG6IW
4SPMWIRS08YGGSPM%1EVMERM0)ZERKIPMWXE1'MXXM0;SSHW/IXEP1MGVS62%WQSHYPEXIXLI
ERKMSKIRMGTVSTIVXMIWSJ,9:)'W&PSSH
*MWL.)7ERXSVS111SVXSR79=Y7=IL6*;]XLI.(IXEPQM6VIKYPEXIWERKMSKIRMG
WMKREPMRKERHZEWGYPEVMRXIKVMX](IZ'IPP
ZER7SPMRKIR'7IKLIVW0&MNOIVO6(YMNW.16SIXIR1/ZER3IZIVIR6MIXHMNO%1IXEP
%RXEKSQMVQIHMEXIH WMPIRGMRK SJ IRHSXLIPMEP GIPP WTIGM½G QMGVS62% MQTEMVW MWGLIQME
MRHYGIHERKMSKIRIWMW.'IPP1SP1IH
;ERK7%YVSVE%&.SLRWSR&%5M<1G%REPP].,MPP.%IXEP8LIIRHSXLIPMEPWTIGM½GQMGVS
62%QM6KSZIVRWZEWGYPEVMRXIKVMX]ERHERKMSKIRIWMW(IZ'IPP
*SRXMNR 6 ,ST ' &VMROQER ,. 7PEXIV 6;IWXIVZIPH% ZER 1SYVMO .% IX EP 1EMRXIRERGI
SJZEWGYPEVIRHSXLIPMEPGIPPWTIGM½GTVSTIVXMIWEJXIVMQQSVXEPM^EXMSR[MXLEREQTLSXVSTLMG
VITPMGEXMSRHI½GMIRXVIXVSZMVYWGSRXEMRMRKLYQERTETMPPSQEZMVYW))(2%)\T'IPP6IW
.EJJI )% 2EGLQER 60 &IGOIV '+ 1MRMGO '6 'YPXYVI SJ LYQER IRHSXLIPMEP GIPPW HIVMZIH
JVSQ YQFMPMGEP ZIMRW -HIRXM½GEXMSR F] QSVTLSPSKMG ERH MQQYRSPSKMG GVMXIVME . 'PMR -RZIWX
ZER;IIP:(IGOIVW11+VMQFIVKIR.1ZER0IYZIR/.0EVHIRS]I.,7GLPMRKIQERR63IX
EP:EWGYPEV IRHSXLIPMEP KVS[XL JEGXSV SZIVI\TVIWWMSR MR MWGLIQMG WOIPIXEP QYWGPI IRLERGIW
Q]SKPSFMRI\TVIWWMSRMRZMZS'MVG6IW
(IZMRI 71:MN 6 6IXXMK 18SHX 0 1G+PEYGLPIR / *MWLIV 2 IX EP 6ETMH QSFMPM^EXMSR SJ
JYRGXMSREPHSRSVLIQEXSTSMIXMGGIPPW[MXLSYX+'7*YWMRK%1(ERERXEKSRMWXSJXLI
'<'67(*MRXIVEGXMSR&PSSH
>LES<5MER(;Y2=MR='LIR.'YM&IXEP8LIWTPIIRVIGVYMXWIRHSXLIPMEPTVSKIRMXSVGIPP
ZME7(*'<'6E\MWMRQMGI.6IGITX7MKREP8VERWHYGX6IW
>IVRIGOI%&MH^LIOSZ/2SIPW,7LEKHEVWYVIR)+ER0(IRIGOI&IXEP(IPMZIV]SJQM
GVS62%F]ETSTXSXMGFSHMIWMRHYGIW'<'0HITIRHIRXZEWGYPEVTVSXIGXMSR7GM7MKREP
VE
1MXGLIPP474EVOMR6//VSL)1*VMX^&6;]QER7/4SKSWSZE%KEHNER]ER)0IXEP'MVGYPEX
MRKQMGVS62%WEWWXEFPIFPSSHFEWIHQEVOIVWJSVGERGIVHIXIGXMSR4VSG2EXP%GEH7GM97%
0E[VMI',+EP7(YRPST,14YWLOEVER&0MKKMRW%44YPJSVH/IXEP(IXIGXMSRSJIPIZEXIH
PIZIPW SJ XYQSYVEWWSGMEXIH QMGVS62%W MR WIVYQ SJ TEXMIRXW [MXL HMJJYWI PEVKI &GIPP P]Q
TLSQE&V.,EIQEXSP
.M<8EOELEWLM6,MYVE=,MVSOE[E+*YOYWLMQE=-[EM24PEWQEQM6EWEFMSQEVOIVSJ
Q]SGEVHMEPMRNYV]'PMR'LIQ
'SVWXIR1*(IRRIVX6.SGLIQW7/Y^RIXWSZE8(IZEY\=,SJWXVE0IXEP'MVGYPEXMRK1M
GVS62%FERH1MGVS62%VI¾IGXQ]SGEVHMEPHEQEKIMRGEVHMSZEWGYPEVHMWIEWI'MVG
'EVHMSZEWG+IRIX
%M.>LERK60M=4Y.0Y=.MES.IXEP'MVGYPEXMRKQMGVS62%EWETSXIRXMEPRSZIPFMSQEVOIV
JSVEGYXIQ]SGEVHMEPMRJEVGXMSR&MSGLIQ&MSTL]W6IW'SQQYR
7ETIX'7MQSRGMRM70SVMSH&4YXLMIV(7EQTSP.2KY]IR'IXEP8LVSQFMRMRHYGIHIR
HSXLIPMEPQMGVSTEVXMGPIKIRIVEXMSRMHIRXM½GEXMSRSJERSZIPTEXL[E]MRZSPZMRK63'/--EGXMZE
XMSRF]GEWTEWI&PSSH
'SQFIW:7MQSR%'+VEY+)%VRSY\('EQSMR07EFEXMIV*IXEP-RZMXVSKIRIVEXMSRSJ
IRHSXLIPMEPQMGVSTEVXMGPIWERHTSWWMFPITVSXLVSQFSXMGEGXMZMX]MRTEXMIRXW[MXLPYTYWERXMGS
EKYPERX.'PMR-RZIWX
&SYPERKIV'1%QEFMPI2+YIVMR%44ERRMIV&0IVS]IV%71EPPEX'2IXEP-RZMZSWLIEV
WXVIWWHIXIVQMRIWGMVGYPEXMRKPIZIPWSJIRHSXLIPMEPQMGVSTEVXMGPIWMRIRHWXEKIVIREPHMWIEWI
,]TIVXIRWMSR
*MGLXPWGLIVIV7(I6SWE7*S\,7GL[MIX^8*MWGLIV%0MIFIXVEY'IXEP'MVGYPEXMRKQMGVS6
2%WMRTEXMIRXW[MXLGSVSREV]EVXIV]HMWIEWI'MVG6IW
>EQTIXEOM%/MIGLP7(VS^HSZ-;MPPIMX41E]V94VSOSTM1IXEP4PEWQEQMGVS62%TVS½P
MRKVIZIEPWPSWWSJIRHSXLIPMEPQM6ERHSXLIVQMGVS62%WMRX]TIHMEFIXIW'MVG6IW
,EVVMW 8% =EQEOYGLM 1 *IVPMXS 1 1IRHIPP .8 0S[IRWXIMR '. 1MGVS62% VIKY
PEXIWIRHSXLIPMEPI\TVIWWMSRSJZEWGYPEVGIPPEHLIWMSRQSPIGYPI4VSG2EXP%GEH7GM97%
=ERK.1EWEEOM-/EQIM2%PIZ'/[SR71/E[EQSXI%IXEP'(GIPPWVITVIWIRXLMKLP]
JYRGXMSREPIRHSXLIPMEPTVSKIRMXSVGIPPWMRQYVMRIFSRIQEVVS[40S73RI I
7XIPPSW/&MKEPOI&0ERKIV,+IMWPIV87GLEH%/SKIP%IXEP)\TVIWWMSRSJWXVSQEPGIPP
HIVMZIHJEGXSVSRGMVGYPEXMRKTPEXIPIXWMWMRGVIEWIHMRTEXMIRXW[MXLEGYXIGSVSREV]W]RHVSQI
ERHGSVVIPEXIW[MXLXLIRYQFIVSJ'(TVSKIRMXSVGIPPW)YV,IEVX.