Vascular Gene Transfer of SDF-1 Promotes Endothelial Progenitor Cell Engraftment and Enhances Angiogenesis in Ischemic Muscle

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1 The Americn Society of Gene & Cell Therpy originl rticle Vsculr Gene Trnsfer of SDF-1 Promotes Endothelil Progenitor Cell Engrftment nd Enhnces Angiogenesis in Ischemic Muscle Michel A Kuliszewski 1, Jeremy Koulnik 1, Jonthn R Lindner 2, Duncn J Stewrt 3 nd Howrd Leong-Poi 1 1 Division of Crdiology, Keenn Reserch Centre in the Li K Shing Knowledge Institute, St Michel s Hospitl, Toronto, Ontrio, Cnd; 2 Division of Crdiovsculr Medicine, Oregon Helth nd Science University, Portlnd, Oregon, USA; 3 The Ottw Hospitl Reserch Institute, University of Ottw, Ottw, Ontrio, Cnd Gene therpy pproches to enhnce endothelil progenitor cell (EPC) homing my ugment cell engrftment to ischemic tissue nd led to greter therpeutic response. Therefore, we ssessed the effects of ultrsound-medited (UM) trnsfection of the chemokine stroml cell derived fctor-1 (SDF-1) on homing nd engrftment of intrvenously dministered EPCs nd the susequent ngiogenic response in chroniclly ischemic skeletl muscle. Bone mrrow derived EPCs were isolted from donor Fisher 344 rts, cultured nd leled in preprtion for injection into recipient nimls vi jugulr vein. Using model of chronic hindlim ischemi in rts, we demonstrted tht UM destruction of intrvenous crrier microules loded with SDF-1 plsmid DNA resulted in trgeted trnsfection of the vsculr endothelium within ischemic muscle nd greter locl engrftment of EPCs. The comintion of SDF-1gene therpy nd EPCs led to the gretest increse in tissue perfusion nd microvsculr density within ischemic muscle, compred to no tretment or either monotherpy lone. Our results demonstrte tht UM trnsfection of SDF-1 improves EPC trgeting to chroniclly ischemic tissue, enhncing vsculr engrftment nd leding to more roust neovsculriztion response. Received 26 Decemer 29; ccepted 23 Jnury 211; pulished online 1 Mrch 211. doi:1.138/mt Introduction Circulting endothelil progenitor cells (EPCs) originting from the one mrrow hve een shown to home to sites of tissue injury, fcilitting endogenous tissue repir, nd vsculr regenertion. 1 Once differentited, ex vivo expnded EPCs hve een reported to integrte into lood vessels nd induce neovsculriztion of ischemic hindlims nd herts in niml models of ischemi nd infrction. 2 Although severl clinicl studies hve now demonstrted the therpeutic potentil of exogenously dministered EPCs to improve left ventriculr ejection frction fter cute myocrdil infrction (MI), 3,4 other studies hve yielded negtive results. 5,6 Given tht ptients t the highest crdiovsculr risk hve the lowest numer nd poorest migrtory nd homing cpcity of endogenous EPCs, the use of utologous EPCs for neovsculriztion in the clinicl setting my prove less effective. 7 In this setting, the ility to increse the homing of exogenously dministered EPCs to specific trget sites my potentilly improve the ngiogenic response to cell therpy. Severl fctors hve een shown to influence EPC moiliztion nd homing to ischemic tissue, including chemokines, 8 ngiogenic cytokines, 9 nd phrmcologic gents. 1 Stroml cell derived fctor-1 (SDF-1) is one such chemokine considered to ply n importnt role in progenitor cell homing nd recruitment for ischemic neovsculriztion. 8,11 We hve previously demonstrted tht ultrsound-medited (UM) destruction of crrier microules ering vsculr endothelil growth fctor (VEGF) plsmid DNA results in improved perfusion within chroniclly ischemic skeletl muscle. 12 Furthermore, this delivery strtegy my e more effective thn direct intrmusculr injections, resulting in directed vsculr trnsfection over wider distriution, leding to more efficient ngiogenic response. 13 In this study, we hypothesized tht UM delivery of microules ering genes encoding for humn SDF-1 would result in loclized trnsfection of the vsculr endothelium nd surrounding myocytes, nd fcilitte mximl homing nd engrftment of intrvenously delivered EPCs, thus cting synergisticlly to promote neovsculriztion of chroniclly ischemic muscle. Results EPC chrcteriztion nd functionl nlysis We first chrcterized the one mrrow derived EPCs used in our experiments, nd confirmed their functionlity fter leling. After 1 dys in culture, EPCs developed n endothelil-like phenotype, expressing endothelil cell surfce mrkers UEA-1, VEGFR-II nd the receptor for SDF-1, CXCR4 (Supplementry Figure S1), with no chnge in cell phenotype fter leling with the fluorophore chloromethyl trimethyl rhodmine (CMTMR) (Supplementry Figure S1,c). After CMTMR leling, cell loss Correspondence: Howrd Leong-Poi, Division of Crdiology, Keenn Reserch Centre in the Li K Shing Knowledge Institute, 6-44 Queen Wing, St Michel s Hospitl, 3 Bond Street, Toronto, Ontrio, Cnd M5B 1W8. E-mil: leong-poih@smh.c Moleculr Therpy vol. 19 no. 5, my

2 SDF-1 gene nd EPC delivery for ngiogenesis The Americn Society of Gene & Cell Therpy Side sctter (log SS) A I 3 J 83.7% 92.9% M.7% Forwrd sctter (log FS) CD34 VEGFR-II CD34/CD133/VEGFR-II 6% % Circulting exogenous EPCs F 4% 2%.7%.4%.2% % Circulting EPCs Endogenous EPCs Nonischemic Ischemic % 5 minutes 3 minutes 6 minutes 24 hours 3 dys 7 dys 14 dys 28 dys.1% Figure 1 In vivo exogenous nd endogenous endothelil progenitor cell (EPC) trcking y fluorescence-ctivted cell sorting (FACS) nlysis in control nd ischemic nimls. () FACS plots showing the cell popultion nd mrker positivity. () Dt (men ± SD) on in vivo exogenous nd endogenous EPC trcking y FACS. Pek exogenous EPC circultion (solid lines left xis) occurred etween 1 nd 7 dys with mximl circultion t dy 3 in oth control nd ischemic groups. The dshed line right xis represents endogenous EPCs. Endogenous EPC levels remined stle throughout the time course of the study. Reltive endogenous mrna expression Reltive endogenous mrna expression Control SDF-1 lone EPCs lone SDF-1 + EPCs Control SDF-1 lone EPCs lone SDF-1 + EPCs c Figure 2 Dt (men ± SD) on stroml cell derived fctor-1 (SDF-1) trnsfection efficcy nd locliztion in hindlim skeletl muscle fter ultrsound-medited trnsfection, nd effects on endogenous SDF-1 expression. Blck rs dy 3, white rs dy 7, gry rs dy 14. () Exogenous (humn) SDF-1 expression y reverse trnscriptse (RT)-PCR in the vrious groups t ll time points (P <.5 versus oth control nd endothelil progenitor cells (EPCs) lone). Mximl trnsgene expression cn e seen t dy 3 in the groups receiving SDF-1 gene therpy. () Endogenous (rt) SDF-1 expression y RT-PCR in the vrious groups t ll time points. There is no difference in endogenous SDF-1 expression long ll groups nd time points. (c) Immunostining for exogenous humn SDF-1. In SDF-1 treted muscle, positive SDF-1 stining (green) is present in the vsculr endothelium of cpillries nd rterioles, with some stining lso evident in the myocytes. No discernle stining is present in control untreted muscle. Br = 5 μm. Blue TO-PRO-3 (nucler counterstin), green humn SDF vol. 19 no. 5 my 211

3 The Americn Society of Gene & Cell Therpy SDF-1 gene nd EPC delivery for ngiogenesis ws miniml, with ~9% cell viility. CMTMR leling did not ffect EPC function in vitro, with similr migrtory cpcity in response to oth SDF-1 nd VEGF nd ngiogenic potentil y mtrigel tuule formtion ssy (Supplementry Figure S1d,e). Time course of EPC circultion Fluorescence-ctivted cell sorting (FACS) nlysis ws used to trck CMTMR-leled EPCs fter intrvenous injection, to determine the time of mximum concentrtion within the circultion. Figure 1 shows the percentge of injected cells tht re circulting t ech time point in control nonligted nimls, nd in nimls fter induction of chronic ischemi (see Animl preprtion). Immeditely fter intrvenous injection (within 3 minutes), very few circulting EPCs were detected. Histologicl nlysis immeditely postinjection demonstrtes tht the mjority of the cells lodge in the lungs. After 24 hours, the numers of cells found in the systemic circultion egin to rise, with cler pek in exogenous circulting EPCs 3 dys postinjection. The presence of chronic ischemi led to miniml chnges in the time course of EPC circultion, with lower circulting numers t dy 3, perhps relted to greter trfficking of EPCs from the circultion into the ischemic muscle. In comprison, there were little chnges in % circulting endogenous EPCs over time (Figure 1). SDF-1 gene trnsfection efficcy nd locliztion Quntittive rel-time reverse trnscriptse (RT)-PCR dt for exogenous SDF-1 mrna from ischemic muscle t vrious time points postdelivery in SDF-1 gene tretment groups is shown in Figure 2. Using specific primers, exogenous trnsgene expression (normlized to the contrlterl nonischemic muscle) ws detected in oth SDF-1 treted nd SDF-1 + EPC treted ischemic muscles, eing similr for oth groups. For oth groups, trnsfection ws gretest t dy 3 postdelivery, decresing over time. The SDF-1 trnsgene ws not detectle in control or EPC lone treted muscle. Endogenous SDF-1 mrna expression is shown in Figure 2, showing no significnt chnges in the groups over time. Immunohistochemicl nlysis for exogenous humn SDF-1 expression ws performed 3 dys postdelivery (Figure 2c). There ws strong positive signl (green stining) in the SDF-1 delivered ischemic muscle, with little to no stining in controls. The mjority of SDF-1 immunostining ws loclized to the endothelium of rterioles nd cpillries with some stining ssocited with myocytes. Time course of EPC engrftment nd cell retention At ech of the study time points EPC engrftment ws ssessed y immunostining. Sections of the proximl hindlim dductor muscle were visulized under confocl microscopy nd cell engrftment/retention ws quntified. EPC retention within the ischemic hindlim dductor muscle t vrious time points fter delivery ws quntified y cell counting. The pek of exogenous retined EPCs occurred t dy 7 postinjection in ll EPC-treted nimls (Figure 3). Mximl exogenous EPC numer ws found in the SDF-1 + EPC group with nerly twofold increse over the EPC lone group (P <.1) t oth dy 7 nd dy 14. There ws no difference in EPC numer in the SDF-1 + EPC nd EPC lone groups t dy 3. Figure 3 illustrtes section of hindlim dductor muscle stined for smooth muscle α-ctin (green), showing severl CMTMR-leled EPCs (red) engrfted within mediumsized rterioles t dy 7 fter delivery within n SDF-1 + EPC treted niml. As expected there ws no detectle exogenous EPC retention in control ischemic hindlims (Figure 4). In the EPC lone treted group (Figure 4), there were numerous cells seen ssocited with cpillries. In the SDF-1 + EPC treted group there were incresed numers of engrfted cells (Figure 4c), predominntly engrfted within smll rterioles nd cpillries within the ischemic hindlim muscle. Muscle perfusion nd vsculr density Perfusion within ischemic dductor muscle in ll groups ws mesured using contrst-enhnced ultrsound (CEU). Representtive CEU-perfusion imges t plteu signl intensity for ll tretment groups prior to nd fter gene delivery re shown in Figure 5. At 2 weeks post-ilic rtery ligtion, immeditely prior to gene delivery, microvsculr lood volume (MBV) ws reduced to ~75 8% nd microvsculr lood flow (MBF) to ~3 35% in the ischemic leg normlized to the contrlterl norml leg in ll four groups. In the control group there ws no chnge in normlized MBV nd MBF over time (Figure 5). After tretment with SDF-1 gene therpy or intrvenous EPCs lone, there were significnt increses EPCs per mm Dys 7 Dys 14 Dys Figure 3 Dt on exogenous endothelil progenitor cell (EPC) engrftment nd retention. () Quntittive dt (men ± SD) on exogenous chloromethyl trimethyl rhodmine (CMTMR)-leled EPC engrftment nd retention in ischemic muscle from ll tretment groups, t multiple time points postdelivery. Dt is expressed s numer of cells per mm 2. Mximl EPC engrftment occurred t 7-dy postinjection in oth groups, with the highest numer of cells seen in the stroml cell derived fctor-1 (SDF-1) + EPC group t oth the 7-dy nd 14-dy time points (P <.1 versus EPCs lone). Blck r EPCs lone, white r EPCs + SDF-1. () Immunostining of medium-sized rterioles t dy 7 postdelivery from the SDF-1 + EPCs group showing exogenously delivered CMTMR-leled EPCs (red stining) ssocited with smooth muscle α-ctin stined rterioles (green stining). Br = 5 μm. Blue = TO-PRO-3 (nucler stin), green smooth muscle α-ctin, red CMTMR-leled exogenous EPCs. Arrow exogenous EPCs. Moleculr Therpy vol. 19 no. 5 my

4 SDF-1 gene nd EPC delivery for ngiogenesis The Americn Society of Gene & Cell Therpy EC lectin stining green CMTMR (+) EPCs red Merge c 2. Normlized microvsculr lood volume # # # # Bseline Dy 3 Dy 7 Dy 14 Figure 4 Representtive imges of exogenous endothelil progenitor cell (EPC) engrftment within ischemic muscle y fluorescent microscopy, t 14-dy postinjection. () No exogenous EPC engrftment in control untreted muscle. () EPC engrftment in the EPC lone treted ischemic muscle. (c) A greter numer of engrfted EPCs in the stroml cell derived fctor-1 + EPC ischemic muscle with engrfted EPCs ssocited with the vessel wlls of cpillries. Br = 5 μm. Blue TO- PRO-3 (nucler stin), green endothelil cell (EC)-lectin, red chloromethyl trimethyl rhodmine (CMTMR) leled exogenous EPCs. Single rrow exogenous EPCs. in normlized MBV nd MBF tht egn t dy 7 postdelivery nd extending to dy 14. In comprison, the improvement in MBF ws significntly greter with comined SDF-1 gene therpy coupled with intrvenous EPC dministrtion, s compred to either monotherpy lone (P <.5 versus SDF-1 lone, nd P <.1 versus EPCs lone, t dys 7 nd 14 fter delivery) (Figure 5). Increses in MBV nd MBF lso occurred t n erlier time point, dy 3, compred to other tretment groups. Fluorescent microngiogrphy (FMA) nlysis reveled reduced vessel density in the control group s ischemic proximl hindlim dductor muscle 4 weeks post-ilic rtery ligtion. Consistent with CEU-perfusion dt, t 14 dys fter gene delivery there were increses in vessel density in ll tretment groups with the gretest increses seen with comintion SDF-1 gene therpy plus intrvenous EPCs (Figure 6). Further FMA nlysis ws performed using rnch ordering to distinguish chnges in cpillry density versus rteriolr density fter therpies (Figure 7). Both SDF-1 lone nd EPC lone treted muscle showed increses in rnch order rtio, indicting higher reltive density of cpillries versus rterioles. In comprison, the comined SDF-1 + EPC group showed restortion to norml rnch order rtio (Figure 7). While oth SDF-1 + EPC nd EPC lone treted ischemic muscle showed significnt increses in cpillry density versus control nontreted muscle (Figure 7c), the comined SDF-1 + EPC treted nimls were the only group tht demonstrted significntly incresed rteriole density t dy 14 postdelivery. Discussion While the success of progenitor cell therpy relies on the ility of cells to repir dmged tissue, it is lso criticlly dependent on. c 1.5 Normlized microvsculr lood flow Control SDF-1 EPCs SDF-1 + EPC # # # # Control SDF-1 EPCs SDF-1 + EPC their homing, migrtion nd retention to sites of injury, regrdless of mode of delivery. The novel finding of this study is tht noninvsive gene- nd cell-sed therpeutic pproch, using vsculr gene trnsfer of SDF-1 y UM destruction of plsmid ering microules to ugment homing nd engrftment of exogenously dministered EPCs, leds to greter ngiogenic response s compred to SDF-1 gene therpy or intrvenous EPCs lone. The physiologic nd pthologic fctors tht influence EPC homing to ischemic tissue remin complex nd poorly understood, with numer of potentil fctors postulted. 14 SDF-1 is chemokine # # # Bseline Dy 3 Dy 7 Dy 14 Figure 5 Proximl hindlim contrst-enhnced ultrsound (CEU) perfusion dt in ll tretment groups. () Representtive color-coded CEU-perfusion imges of ischemic hindlim muscle lood flow from ll groups t 14-dy postdelivery. CEU signl from microules ws gretest for comined stroml cell derived fctor-1 (SDF-1) + endothelil progenitor cell (EPC)-treted ischemic muscle. () Microvsculr lood volume (men ± SD) in the ischemic muscle, normlized to contrlterl nonischemic muscle for the four tretment groups; t seline (pretretment) nd t 3, 7, nd 14 dys postdelivery. P <.1 compred to corresponding dt in control untreted nimls, # P <.5 compred to corresponding seline (predelivery) dt. (c) Microvsculr lood flow (men ± SD) in the ischemic muscle, normlized to contrlterl nonischemic muscle for the four tretment groups; t seline (pretretment) nd t 3, 7, nd 14 dys postdelivery. P <.5 compred to corresponding dt in control untreted nimls, # P <.5 compred to corresponding seline (predelivery) dt, P <.5 compred to corresponding dt in SDF-1 treted nimls, P <.1 compred to corresponding dt in EPC-treted nimls vol. 19 no. 5 my 211

5 The Americn Society of Gene & Cell Therpy SDF-1 gene nd EPC delivery for ngiogenesis c Numer of vessels per mm 2 3, 2,5 2, 1,5 1, 5 Norml leg Control SDF-1 lone EPCs lone SDF-1 + EPCs Figure 6 Dt on proximl hindlim vsculr density in ll tretment groups. () Representtive stcked fluorescent microngiogrphic (FMA) imges of microvessels in ischemic hindlim muscle fter no tretment (control), ultrsound-medited delivery of stroml cell derived fctor-1 (SDF-1) lone, exogenous endothelil progenitor cells (EPCs) dministrtion nd comintion of SDF-1 gene delivery nd exogenous EPC delivery. Br = 5 μm. () Computerized rendering of the ove imge stcks, from which vsculr density is quntified. Note the greter vsculr density in the SDF-1 + EPC group versus ll other groups. (c) Quntittive microvessel density y FMA (men ± SD) of ischemic hindlim muscle t dy 14 postdelivery, in ll tretment groups. At 14-dy postdelivery, FMA reveled significnt increse in the density of microvessels in the ischemic leg of oth EPC delivery groups, with mximl vessel density occurring in the SDF-1 + EPC group. P <.5 compred to corresponding dt from control ischemic muscle. considered to ply n importnt role in hemtopoietic stem cell trfficking, 11 nd hs een shown to ply criticl role in stem cell recruitment for ischemic neovsculriztion. Severl studies hve investigted the therpeutic role of SDF-1 for endogenous EPC recruitment nd repir. In mouse model of coronry ligtion, gene delivery of SDF-1 using n denovirl vector fter cute MI more thn douled endogenous one mrrow derived EPC recruitment into ischemic myocrdium. 8 Similrly, gene trnsfer of SDF-1 promotes EPC moiliztion nd homing for ischemic neovsculriztion vi VEGF/endothelil nitric oxide synthse relted pthwys. 15 Finlly, SDF-1 protein dministrtion into wounds reversed the nitric oxide medited EPC homing impirment in dietes, enhncing wound heling. 16 This study suggests tht SDF-1 could potentilly e used to overcome the functionl impirment of EPCs in disese sttes tht my limit their therpeutic pplictions. 7 In the only study tht exmined the effects of SDF-1 on the therpeutic potentil of exogenously dministered EPCs, Ymguchi et l. 17 performed intrmusculr injections of SDF-1 protein comined with intrvenous humn EPC trnsplnttion into the cutely ischemic hindlim muscle of thymic nude mice. They found n incresed locl ccumultion of fluorescence-leled EPCs within ischemic muscle in the SDF-1 tretment group, which ws ssocited with n increse in tissue perfusion nd cpillry density. We hve now extended this concept, demonstrting tht comined gene- nd cell-sed therpy pproch, using SDF-1 gene delivery in conjunction with intrvenous EPC dministrtion, results in n enhnced therpeutic neovsculriztion in the setting of chronic ischemi, compred to either therpy lone. The sl incorportion rte of exogenous EPCs into norml tissue, in the sence of injury, is extremely low. 18 EPC engrftment is enhnced in the setting of tissue injury or ischemi, with engrftment eing greter in the setting of cute s compred to chronic 4 c 12, P <.5 d 12, P <.5 Brnch order Norml Control SDF-1 leg lone Cpillry density (mm 3 ) # # # 6, EPCs SDF-1 + lone EPCs Norml leg Control SDF-1 lone EPCs lone # # SDF-1 + EPCs Arteriolr density (mm 3 ) 6, Norml leg Control SDF-1 lone # EPCs lone # SDF-1 + EPCs Figure 7 Dt (men ± SD) on rnch order, cpillry nd rteriolr density of ischemic hindlim muscle t dy 14 postdelivery from ll groups, y fluorescent microngiogrphy nd 3D rnch order nlysis using the Strhler Method. () Representtive color-coded imges of rteriolr nd cpillry density from hindlim skeletl muscle in norml rts, nd ischemic rts from ll four groups, t dy 14 postdelivery. Red rnch order 1 3 (rterioles), white rnch order (cpillries). () Both stroml cell derived fctor-1 (SDF-1) nd endothelil progenitor cells (EPCs) lone groups hd increses in rnch order rtio, indicting reltively higher density of cpillries:rterioles. The comined SDF-1 + EPC treted ischemic muscles showed restortion to norml rnch order. (c) The comined SDF-1 + EPC nd EPC lone treted ischemic muscles showed significnt increses in cpillry density versus control, (d) while the comined EPC +SDF-1 group ws the only tretment group tht demonstrted significntly incresed rteriolr density compred to control untreted nimls. P <.5 versus norml leg, # P <.5 versus control. Moleculr Therpy vol. 19 no. 5 my

6 SDF-1 gene nd EPC delivery for ngiogenesis The Americn Society of Gene & Cell Therpy ischemi. 19 Thus, strtegies to enhnce homing nd engrftment would e vlule for improving the therpeutic effect of EPCsed therpies, prticulrly in the setting of chronic ischemi, s in our study. In the mjority of studies to dte, exogenous EPC delivery hs een vi locl dministrtion, minly intrmusculr nd intrcoronry injections. These reltively invsive techniques mke them less suitle for repeted dministrtions. The ility to use n intrvenous dministrtion of EPCs would e n ttrctive, noninvsive technique to deliver EPCs. However, studies hve shown lck of trgeting with this method, with the solute level of rdioleled EPCs homing to the norml hert eing ~1% fter intrvenous injection. 2 This increses pproximtely twofold to 2% fter cute MI, strong stimulus for homing nd engrftment. When EPCs re injected intr-rterilly, however, the engrftment rte is significntly higher, ~4.7-fold, fter cute MI. 2 Dt from humn studies hve confirmed these findings. After intrcoronry dministrtion of rdioleled EPCs, the verge ctivity within the first 24 hours ws highest mong ptients with cute MI, nd progressively decresing with time from infrction, with lowest ctivity fter intrcoronry injection seen in ptients with chronic (>1 yer post) MI. This mrked difference in EPC engrftment nd retention in the setting of cute versus chronic ischemi is in keeping with studies demonstrting tht the chemotctic fctors VEGF nd SDF-1 re downregulted in chronic ischemi s opposed to upregulted in more cute ischemi. 21 Thus, method to increse trgeting nd engrftment of intrvenously delivered EPCs, specificlly in the setting of chronic ischemi or infrction where endogenous homing signls re low, would e n importnt first step in developing noninvsive method of EPC delivery for therpeutic ngiogenesis. An importnt spect of our study ws the determintion of the time course of systemic EPC circultion fter n intrvenous injection in norml nd chroniclly ischemic nimls, which ws not exmined in the prior study of comined SDF-1 protein nd EPC delivery. 17 Using FACS, we were le to trck our CMTMR-leled EPCs, showing tht immeditely fter injection into the jugulr vein there were very few detectle exogenous EPCs in the peripherl circultion, due predominntly to lodging nd retention within the lungs. 22 After initil pulmonry retention, exogenous EPCs slowly reppered in the peripherl circultion, eginning t 24 hours, nd peking t dy 3 postinjection, eventully tpering off y dy 14. In the presence of chronic ischemi, the time course of EPC circultion remined unchnged. We susequently designed our delivery protocols to llow for pek SDF-1 gene trnsfection to coincide with mximl systemic circultion of our exogenously dministered EPCs. We hve previously shown tht mximl gene trnsfection fter UM microule destruction occurs t dy 3 postdelivery. 12,13 This llowed us to dminister intrvenous EPCs simultneously with UM SDF-1 plsmid DNA delivery. Gene trnsfer strtegies yield longer durtion of therpeutic ction thn loclized protein delivery, s noted in our study where SDF-1 trnsgene expression ws detectle out to 14 dys postdelivery, thus providing continuous homing signl from our trgeted ischemic tissue for the full durtion of EPC circultion fter intrvenous dministrtion. Another dvntge of our technique is the specific site of trgeted gene delivery. As compred to direct intrmusculr injections, UM gene delivery results in directed vsculr trnsfection over wider distriution, leding to more effective gene delivery strtegy. 13 Similr to our previous studies, 12,13 SDF-1 gene trnsfection y UM destruction of DNA-ering microules ws seen to loclize to the vsculr endothelium nd surrounding myocytes, where ugmenttion of signling for circulting EPC homing nd trnsmigrtion my e fully mximized. In our study, the vsculr gene trnsfer of SDF-1 more thn douled exogenous EPC engrftment into the vsculture of ischemic hindlim muscle out to 14 dys fter delivery, resulting in n enhnced ngiogenic response, greter density of neovessels nd increses in oth rteriolr nd cpillry density while preserving norml rnch order rtios. Increses in tissue perfusion were evident s erly s 3 dys postdelivery in our comined SDF-1 plus EPC treted nimls s compred to EPC tretment lone, despite similr numers of engrfted EPCs t tht erly time point. This finding is in keeping with dt suggesting tht ctivtion of the SDF-1/CXCR4 xis enhnces collterl flow, t lest in prt, vi direct effects on vsculr endothelil cells. 23 Our study hs severl importnt limittions. While we were le to follow the engrftment of our exogenous CMTMR-leled EPCs, we were unle to trck endogenous EPCs tht my lso home to sites of SDF-1 gene trnsfer. Thus we cnnot determine the reltive effects of exogenous versus endogenous EPC engrftment on the neovsculriztion response to SDF-1 gene therpy. Regrdless, the ddition of exogenous EPCs to SDF-1 gene therpy resulted in further increment over SDF-1 therpy lone, suggesting n importnt contriution y cell trnsplnttion. We do not hve ny dt on long term EPC engrftment, eyond 14 dys fter delivery. We did not study the effect of microule destruction lone on progenitor cell recruitment, s hs een previously studied. 24 In our prior study of UM gene delivery, destruction of microules ering control plsmid DNA did not result in upregultion of endogenous ngiogenic genes or restortion in hindlim perfusion s compred to no tretment, suggesting lck of significnt iologic effect. 12 In summry, UM trnsfection of SDF-1 improves intrvenous EPC trgeting to ischemic tissue, enhncing vsculr EPC engrftment into chroniclly ischemic tissue nd leding to greter neovsculriztion response. Strtegies for comined gene- nd EPC-sed therpies my e more effective thn either therpy lone, for promoting ngiogenesis nd improving perfusion to chroniclly ischemic tissue. Mterils nd Methods Animl preprtion. The study protocol ws pproved y the Animl Cre nd Use Committee t St Michel s Hospitl Reserch Centre, University of Toronto (Toronto, Ontrio, Cnd). Proximl hindlim dductor muscle ischemi ws produced in 126 nesthetized Fisher F344 rts. Under sterile conditions, the left common ilic rtery nd smll proximl rnches were exposed nd ligted with 4 silk sutures. The incision ws closed in lyers nd nimls were recovered. In this model, while flow is immeditely reduced to ~25% norml, endogenous ngiogenesis occurs over the susequent 2 weeks, fter which perfusion remins chroniclly reduced in the proximl dductor muscles t ~4 5% of norml, 12,13,25 without lim necrosis or uto-mputtion. Cell preprtion nd leling. EPCs used for ll our experiments were isolted from the tiis nd femurs of 3 5-week-old syngeneic Fisher 344 rts (Chrles River Lortories, Sherrooke, Queec, Cnd). The spirted mrrow ws centrifuged nd one mrrow cells plted on fironectin coted flsks t density of >1 1 6 cells/ml nd grown in supplemented endothelil cell sl medium 2 (Cedrlne Lortories, 9 vol. 19 no. 5 my 211

7 The Americn Society of Gene & Cell Therpy SDF-1 gene nd EPC delivery for ngiogenesis Burlington, Ontrio, Cnd) for 1 dys, to promote differentition into n endothelil phenotype. 26 EPCs were leled using the vile fluorophore CMTMR (Invitrogen, Burlington, Ontrio, Cnd), which provides n ccurte method of detecting ex vivo leled cells out to severl months. 22 The cells were llowed to recover for 24 hours fter leling, efore eing injected (see Supplementry Mterils nd Methods for further detils). EPC chrcteriztion nd functionl nlysis. Unleled nd CMTMRleled EPCs were stined for the presence of mture endothelil mrkers, endothelil specific lectin, UEA-1 (1:2) (Sigm, Okville, Ontrio, Cnd), VEGFR-II (1:5) (R&D Systems, Minnepolis, MN) nd CXCR4 (1:15) (cm, Cmridge, MA). Percent mrker positivity ws determined using FACS nlysis. In vitro EPC function ws ssessed y stndrd Boyden Migrtion nd Mtrigel Tuule formtion ssys 27 (see Supplementry Mterils nd Methods for further detils). Time course of exogenous EPC circultion. The time course of circulting exogenous EPCs fter n intrvenous injection ws studied in 12 rts, six control nonligted rts, nd six rts t 2 weeks fter induction of hindlim ischemi y left ilic rtery ligtion. CMTMR-leled EPCs (1 1 6 in 1 ml sterile sline) were injected vi the right jugulr vein over 1 minute. Blood smples (1 µl) were serilly tken t 5, 3, nd 6 minutes for short-term trcking. For longer term trcking 1 µl smples were tken t 1, 3, 7, 14, nd 28 dys postinjection. Whole lood smples were lysed with BD FACS lysing solution (BD Biosciences, Mississug, Ontrio, Cnd), nd nlyzed y FACS to quntify circulting CMTMR-leled EPCs, s well s endogenous non-cmtmr-leled EPCs using specific EPC mrkers VEGFR-II, CD34 nd CD133 (see Supplementry Mterils nd Methods for further detils). Microule nd DNA preprtion nd ssemly. Microules with ctionic lipid shell were creted s previously descried. 12,13 For perfusion imging, lipid-shelled decfluoroutne microules were used. Microule concentrtions were determined using Coulter Multisizer IIe (Beckmn-Coulter, Mississug, Ontrio, Cnd), prior to intrvenous dministrtion (see Supplementry Mterils nd Methods for further detils). Perfusion imging. CEU imging of the proximl hindlim dductor muscles ws performed s previously descried, 12,13,25,27,28 to otin perfusion dt on MBV, velocity, nd MBF 12,28 (see Supplementry Mterils nd Methods for further detils). Gene delivery. For UM gene delivery, ultrsound trnsmission ws performed with phsed rry trnsducer (Sonos 55, Philips Ultrsound) t 1.3 MHz nd trnsmit power of.9w (12 V, 9 ma), using pulsing intervl of 5 seconds. Ctionic microules (1.5 ml; ) chrgecoupled with 5 μg of humn SDF-1 cdna (InvivoGen, Sn Diego, CA) ws infused over 1 minutes intrvenously. To llow wider field of delivery, ultrsound ws trnsmitted during slow sweep long the length of the proximl ischemic hindlim, for totl of 2 minutes 12,13 (see Supplementry Mterils nd Methods for further detils). FMA. FMA, method of postmortem vsculr csting, ws performed to quntify vessel density, s previously descried. 12,13 Cpillry nd rteriolr density ws clculted y rnch order nlysis sed on the Strhler Method, 29 using Neurolucid Softwre pckge (MBF Bioscience, Williston, VT) (see Supplementry Mterils nd Methods nd Supplementry Figure S2 for further detils). Immunohistochemistry. In vivo EPC engrftment nd sptil locliztion were determined using immunohistochemistry. Cryo-locks of explnted tissue were sectioned (15 μm thick) every 25 μm nd rehydrted, fixed in 2% prformldehyde (Sigm) in phosphte-uffered sline, nd wshed. Cell surfce ntigens were identified using: mouse nti-humn CD31 (Cedrlne Lortories), rit nti-humn SDF-1 (Upstte Biotechnology, Wlthm, MA), mouse nti-rt α-ctin (cm) nd endothelil cell specific lectin (UEA-1) (Sigm). The presence of ntiody ws confirmed y exposure to phycoerythrin or fluorescein isothiocynte conjugted secondry ntiody. TO-PRO-3 (Invitrogen) ws used s nucler mrker (see Supplementry Mterils nd Methods for further detils). RT-PCR. Quntittive rel-time RT-PCR for exogenous SDF-1 trnscript nd endogenous SDF-1 were performed, using previously descried methods 12,13 (see Supplementry Mterils nd Methods for further detils). Experimentl protocol. CEU-perfusion imging of the ischemic nd contrlterl control hindlim dductor muscles ws performed 14 dys fter ligtion, t time when endogenous ngiogenesis ws complete. Delivery ws then performed, ccording to ssigned tretment groups: group 1: control group, no tretment; group 2: UM SDF-1 plsmid microule delivery; group 3: intrvenous EPC delivery; group 4: SDF-1 plsmid microule delivery plus intrvenous EPC delivery (n = 3 per group). CEU imging ws performed t dys 3, 7, nd 14 postdelivery (n = 1 per group). In five rts per group (dy 14), FMA ws performed efore eing killed. Tissue for immunohistochemistry nd RT-PCR ws otined from the remining rts ischemic nd nonischemic hindlims, lungs, hert, nd liver. Sttisticl methods. Dt re expressed s men ± SD. Comprisons of dt recorded t single time point ws performed y two-tiled unpired Student t-tests tht ssumed unequl vrince. Anlyses of dt cquired t severl time points for multiple groups were performed y two-wy nlysis of vrince; if significnt, Bonferroni correction for multiple comprisons ws pplied when post hoc nlysis etween different time points or etween different groups t the sme time point ws performed. SUPPLEMENTARY MATERIAL Figure S1. Phenotypic chrcteriztion nd functionl nlysis of endothelil progenitor cells (EPCs), efore nd fter CMTMR leling. Figure S2. Color-coded digrm illustrting rnch order nlysis sed on the Strhler Method. Mterils nd Methods. ACKNOWLEDGMENTS This work ws supported y n Operting Grnt (FRN 62763) from the Cndin Institutes of Helth Reserch, Ottw, Ontrio, Cnd, nd n Equipment Grnt from the Cndin Foundtion for Innovtion, Ottw, Ontrio, Cnd. J.R.L. is supported y grnts R1-HL-74443, R1-HL-7861, nd R1-DK-6358 from the Ntionl Institutes of Helth, Bethesd, MD. H.L.-P. is supported y Phse II Clinicin Scientist Awrd, Hert nd Stroke Foundtion of Ontrio, Ottw, Ontrio, Cnd nd n Erly Resercher Awrd from the Ministry of Reserch nd Innovtion, Ontrio, Cnd. The uthors declred no conflict of interest. REFERENCES 1. Ashr, T, Msud, H, Tkhshi, T, Klk, C, Pstore, C, Silver, M et l. (1999). 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