integrative physiology - circulation...

Therapeutic Arteriogenesis by Ultrasound-MediatedVEGF165 Plasmid Gene Delivery to Chronically Ischemic

Skeletal MuscleHoward Leong-Poi, Michael A. Kuliszewski, Michael Lekas, Matthew Sibbald,

Krystyna Teichert-Kuliszewska, Alexander L. Klibanov, Duncan J. Stewart, Jonathan R. Lindner

Abstract—Current methods of gene delivery for therapeutic angiogenesis are invasive, requiring either intraarterial orintramuscular administration. A noninvasive method of gene delivery has been developed using ultrasound-mediateddestruction of intravenously administered DNA-bearing carrier microbubbles during their microcirculatory transit. Herewe show that chronic ischemia could be markedly improved by ultrasound-mediated destruction of microbubblesbearing vascular endothelial growth factor-165 (VEGF165) plasmid DNA. Using a model of severe chronic hindlimbischemia in rats, we demonstrated that ultrasound mediated VEGF165/green fluorescent protein (GFP) plasmid deliveryresulted in a significant improvement in microvascular blood flow by contrast-enhanced ultrasound, and an increasedvessel density by fluorescent microangiography, with minimal changes in control groups. The improvement in tissueperfusion was attributed predominantly to increases in noncapillary blood volume or arteriogenesis, with perfusionpeaking at 14 days after delivery, followed by a partial regression of neovascularization at 6 weeks. Transfection waslocalized predominantly to the vascular endothelium of arterioles in treated ischemic muscle. RT-PCR confirmed thepresence of VEGF165/GFP mRNA within treated ischemic muscle, being highest at day 3 postdelivery, and subsequentlydecreasing, becoming almost undetectable by 6 weeks. We found a modulation of endogenous growth factor expressionin VEGF-treated ischemic muscle, consistent with a biologic effect of ultrasound mediated gene delivery. The resultsof our study demonstrate the utility of ultrasonic destruction of plasmid-bearing microbubbles to induce therapeuticarteriogenesis in the setting of severe chronic ischemia. (Circ Res. 2007;101:295-303.)

Key Words: angiogenesis � gene therapy � contrast ultrasound � peripheral vascular disease� chronic ischemia

In the setting of severe coronary artery disease (CAD) andperipheral arterial disease (PAD), endogenous neovascu-

larization represents the body’s most important attempt torestore tissue perfusion toward normal. These responses,however, are often inadequate to prevent debilitating symp-toms and ischemic tissue loss. Because of the need for someform of immediate palliative therapy, there have been numer-ous clinical trials designed to promote new vessel growth byexogenous administration of proangiogenic genes in patientswith refractory ischemic symptoms and end-organ damage.1,2

Although initial small open-labeled trials yielded promisingresults, subsequent larger double-blind randomized placebo-controlled clinical trials have failed to show much clinicalbenefit.3–5 The largely disappointing results of clinical trialsof therapeutic angiogenesis may in part be explained bysuboptimal delivery of genetic material to target cells ortissue.6,7 Intraarterial delivery of genes is largely ineffective

because of insufficient amounts of transfection, and can alsoresult in systemic delivery to nontargeted tissue.8 Althoughintramuscular delivery has been shown to more effective,8

this strategy is still limited by the inability to target certaincell types, the relatively localized nature of transfection inproximity to the injection site, and the impracticality forrepetitive treatments in tissues such as the heart. The ability tononinvasively deliver genetic material to specific targettissues, such as the vascular endothelium, in a controlledmanner would be an important step toward a safe andeffective proangiogenic gene therapy.

A noninvasive gene delivery strategy has been developedusing ultrasound-mediated destruction of intravenously (i.v.)administered DNA-bearing carrier microbubbles during theirmicrocirculatory transit.9 This strategy has been used toamplify transfection of reporter plasmid DNA to skeletal10

and cardiac muscle.11 The mechanism by which gene trans-

Original received November 21, 2006; resubmission received January 17, 2007; revised resubmission received May 21, 2007; accepted June 7, 2007.From the Division of Cardiology (H.L.P., M.A.K., M.L., M.S., K.T.K., D.J.S.), Keenan Research Centre in the Li Ka Shing Knowledge Institute, St

Michael’s Hospital, Toronto, Ontario, Canada; the Cardiovascular Division (J.R.L.), Oregon Health and Science University, Portland; and the Universityof Virginia (A.L.K.), Charlottesville.

Correspondence to Howard Leong-Poi, MD, 7-052 Bond Wing, St Michael’s Hospital, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8. [email protected]

© 2007 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.107.148676

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fection is enhanced likely involves extravascular depositionof DNA by either transient microporation produced by highvelocity microjets during gas volume oscillation in theultrasound field, or overt microvascular rupture.9,10 Recently,this strategy has been used to deliver plasmid encoding humanhepatocyte growth factor (HGF) to infarcted hearts in rats,12 andto deliver vascular endothelial growth factor (VEGF) to normalrat myocardium.13

In this study, we hypothesized that that VEGF165 transfec-tion by ultrasonic destruction of plasmid DNA-bearing mi-crobubbles administered intravenously would improve skel-etal muscle perfusion in the setting of severe ischemic PAD.The study was designed to test whether this vascular-basedstrategy for proangiogenic gene therapy would be effective inthe setting of reduced limb blood flow and whether therapywould be additive to native neovascularization. A secondaryaim of our study was to determine whether gene deliverycould be localized to the vasculature where therapy withVEGF165 is likely to be most effective.

Materials and MethodsMicrobubble and DNA PreparationPlasmid DNA was charge-coupled to cationic lipid microbubbles aspreviously described.10 Microbubbles with a cationic (zeta potentialof �60 mV) lipid shell14 were created, which when incubated withplasmid DNA, results in approximately 6700 plasmids on surface ofeach microbubble.10 For perfusion imaging, nontargeted lipid-shelled decafluorobutane microbubbles (MP1950) were used. Micro-bubble concentrations were determined by electrozone sensing witha Coulter Multisizer IIe (Beckman-Coulter).

Plasmid vectors were constructed for transfection of enhancedgreen fluorescent protein (GFP) alone or the cotransfection of bothhuman VEGF165 and GFP. For the latter, we constructed a bicistronicvector encoding human VEGF165 and GFP, which incorporates aninternal ribosome entry site (IRES) that facilitates the translation ofboth proteins from a single mRNA molecule, with high levels ofexpression of both genes within the same cell.15

Animal PreparationThe study protocol was approved by the Animal Care and UseCommittee at St Michael’s Hospital-Health Sciences Research Cen-tre, University of Toronto. Proximal hindlimb ischemia was pro-duced in 90 Sprague-Dawley rats. Rats were anesthetized withintraperitoneal injection of ketamine hydrochloride (10 mg�kg�1) andxylazine (8 mg�kg�1). Using aseptic technique, the left common iliacartery and small proximal branches were exposed and ligated with4-0 suture. The incision was closed in layers and animals wererecovered.

Perfusion ImagingContrast-enhanced ultrasound (CEU) imaging of the proximal hind-limb adductor muscles was performed with gated pulse inversionimaging (HDI 5000, Philips Ultrasound) at a mechanical index of 1.0and a transmission frequency of 3.3 Mhz (L7-4 transducer). Gainsettings were optimized and held constant. Data were recorded onmagnetic-optical disk and transferred to a computer for off-lineanalysis.

Perfusion in the adductor muscles was assessed during continuousi.v. infusion of nontargeted MP1950 microbubbles (1�107 min�1).Background images were acquired at baseline for subtraction oftissue signal. Intermittent imaging was then performed by progres-sive prolongation of the pulsing interval (PI) from 0.2 to 20 s, usingthe internal timer. Several averaged background frames were digi-tally subtracted from averaged contrast-enhanced frames at each PI.PI versus signal intensity (SI) data were fit to the function, y�A(1�e��t), where y is SI at the pulsing interval t, A is plateau video

intensity which is an index of microvascular blood volume (MBV),and � is the rate constant which provides a measure of microvascularblood velocity.16 Microvascular blood flow (MBF) was calculated bythe product of A and �.

CEU perfusion data were recalculated using averaged frames at aPI of 1 s as background to eliminate signal from vessels with atransaxial plane velocity of 2.4�10�3 m/s.17 This process eliminatessignal from almost all noncapillary microvessels, with minimal lossof capillary signal, thereby yielding information from the capillarycompartment alone. The relative noncapillary blood volume (NCBV)was determined by the difference of the calculated A-values. Thisalgorithm has been previously used to measure dynamic changes inskeletal muscle capillary volume, and has been validated againstassays for capillary xanthine oxidase availability.17

Ultrasound-Targeted Gene DeliveryFor gene delivery, ultrasound transmission was performed with aphased array transducer (Sonos 5500, Philips Ultrasound) at 1.3MHz using B-mode ultraharmonic imaging at a transmit power of0.9W (120 V, 9 mA). Cationic microbubbles (1x109) coupled with500 �g of cDNA were infused intravenously over 10 minutes. Toallow for a wider field of delivery, the transducer was positionedtransverse to the ischemic adductor muscles and ultrasound wastransmitted during a slow sweep along the length of the proximalhindlimb muscles. A PI of 5 s was used to allow microbubblereplenishment into the beam elevation between each pulse ofultrasound. Ultrasound transmission was continued for 10 minutesafter cessation of the infusion, to destroy remaining circulatingDNA-loaded microbubbles.

Fluorescent MicroangiogaphyBefore sacrifice, the abdominal aorta was cannulated and the distalhindlimbs flushed with 20 mL of heparinized saline. A 10% solutionof fluorescent microspheres (2 �m diameter; Sigma) mixed with a1% solution of low melting point agarose at 45°C was slowlyinjected into the aortic cannula. The animal was euthanized andplaced in an ice bath to facilitate rapid cooling and solidification ofthe casting agent. Hindlimb muscle was removed and placed in 10%buffered formalin and sectioned (200 �m). Using confocal micros-copy, a series of stacked images (4-�m slices) was taken and themiddle 25 slices (100 �m total thickness) were projected to quantifythe density of blood vessels using automated software (IPTKanalysis software, Reindeer Graphics Inc). This technique has beenpreviously described to quantify pulmonary vascular density inexperimental models of pulmonary arterial hypertension.18

ImmunohistochemistryIn vivo transfection efficacy and spatial localization was determinedusing immunohistochemistry (see Methods supplement for specificdetails on tissue processing techniques, available online at http://circres.ahajournals.org). Cell surface antigens were identified using:mouse anti-human CD31 (Alpha Diagnostics Intl Inc), mouse anti-human Tie-2 (Clone Ab33, Upstate Biotechnology), UEA-1 (Sigma),and mouse anti-human Alpha-actin (Sigma). The presence of anti-body was confirmed by exposure to a phycoerythrin (PE) conjugatedsecondary antibody. TOPO-3 (Sigma) was used as a nuclear marker.

RT-PCRSemi-quantitative real-time RT-PCR for endogenous rat VEGF,angiopoietin-1 (Ang-1), and Angiopoietin-2 (Ang-2) mRNA, as wellas exogenous GFP and VEGF165/GFP transcripts were performedusing standard techniques in our laboratory (see online methodssupplement for further details on tissue processing and specificprimers used).

Western BlottingWestern blotting was performed to measure total VEGF165 proteinlevels in ischemic hindlimb muscle at various time points postligation in the VEGF-treated group, using standard techniques in ourlaboratory (see online methods supplement for specific details).

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Experimental ProtocolCEU perfusion imaging of both hindlimb adductor muscles wasperformed 14 days after iliac artery ligation. Ultrasound-targetedgene delivery was then performed, according to assigned treatmentgroup: group 1: control, no treatment; group 2: GFP plasmid; group3: VEGF165/GFP plasmid (n�18 per group). Repeat CEU wasperformed at days 17 (n�6 per group) and 28 (n�12 per group). Toassess late gene transfection and efficacy, an additional 36 rats(n�12 per treatment group), were studied at 8 weeks after ligation.In 4 rats per group, fluorescent microangiogaphy (FMA) wasperformed immediately before sacrifice. In remaining animals,skeletal muscle tissue from the ischemic and nonischemic adductormuscles, as well as tissue from the lungs, heart, and liver wasobtained for postmortem immunohistochemistry, quantitative RT-PCR, and Western blotting. Normal hindlimb muscles from anadditional 6 animals without ligation were obtained for semiquanti-tative RT-PCR, for comparison to hindlimb muscles from ischemicanimals that underwent ligation.

Statistical MethodsData are expressed as mean�SD. Comparisons between multiplestages were made with 1-way ANOVA. When differences werefound, interstage comparisons were performed using nonpairedStudent t test with Bonferroni correction. Data for pre- and postgenetherapy were compared by paired Student t test. Differences wereconsidered significant at P�0.05 (2-sided).

ResultsMuscle Perfusion and Vascular DensityTwo weeks after iliac artery ligation, blood flow to theischemic adductor muscles was reduced to approximately�40% of normal (Figure 1). No changes in MBV or MBF

were seen at 3 days postdelivery (day 17 postligation) in anygroup. In group 1 control rats, there was no change in MBVor MBF over the subsequent 8 weeks. In group 2 ratsreceiving microbubbles bearing GFP-plasmid alone, therewas a small but significant increase in MBV at 2 weekspostdelivery, but no change in MBF. At 8 weeks, MBV hadreturned to pretreatment levels. In comparison, group 3 ratsreceiving microbubbles bearing VEGF165/GFP plasmidshowed significant improvements in both MBV and MBF 2weeks after ultrasound-mediated gene delivery. By 8 weeks,MBV and MBF had decreased, however remained greaterthan baseline pretreatment values (Figure 1). Examples ofCEU perfusion imaging of the ischemic muscle from each ofthe treatment groups are illustrated in Figure 2.

Image processing algorithms were applied to differentiatetotal microvascular flow from capillary blood flow (CBF).This analysis demonstrated that GFP-plasmid microbubbledelivery increase MBV primarily by increasing noncapillaryblood volume (NCBV; Figure 3), which did not translate toincreases in CBF or MBF. Ultrasonic destruction of micro-bubbles bearing VEGF165/GFP plasmid resulted in a greaterincrease in NCBV, which was associated with a normaliza-tion of CBF (Figure 3). Similar to total blood flow, there waspartial regression of NCBV in VEGF165/GFP muscle by 8weeks postligation.

FMA demonstrated reduced vessel density in nontreatedischemic muscle 28 days and 8 weeks after iliac arteryligation (Figure 4). In keeping with CEU data, there was a

Figure 1. Microvascular blood volume (MBV; A)and microvascular blood flow (MBF; B) in theischemic muscle, normalized to contralateralnonischemic muscle for the 3 treatment groupsat 14 days (pretreatment), 17 days, 28 daysand 8 weeks postligation. Ultrasound-mediateddelivery of VEGF165 to ischemic muscles pro-duced significant increases in normalized MBVand MBF peaking at day 28, with partialregression at 8 weeks, whereas nontreatedcontrol ischemic muscles remained unchanged.GFP-treated muscle had minor increases inMBV at day 28, without significant changes inMBF. *P�0.01 compared with correspondingdata in control animals, †P�0.005 comparedwith corresponding data in GFP-treated ani-mals, ‡P�0.01 compared with corresponding14 day data.

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minor increase in vessel density in GFP plasmid–treatedmuscle at day 28, which was not present at 8 weeks. InVEGF165-treated ischemic muscle, vessel density at day 28was significantly greater than both untreated and GFP-plasmid treated ischemic muscle (Figure 4), with partialregression at 8 weeks.

Efficacy of Gene TransfectionAt day 17 (3 days postdelivery), a strong GFP signal wasdetected only in plasmid-treated ischemic muscle, with noGFP signal in control untreated ischemic muscle (Figure 5A).Although this signal was predominantly localized to thevascular endothelial layer of small to medium sized arterioles

Figure 2. Representative contrast-enhanced ultra-sound (CEU) perfusion images of ischemic hind-limb muscle at increasing pulsing intervals (PI),and the corresponding PI vs signal intensity curvesfrom 1 animal in each of the 3 treatment groupsat 4 weeks postligation. Contrast enhancementinto ischemic skeletal muscle was greater andoccurred faster in VEGF165/GFP-treated musclecompared with both GFP-treated and untreatedischemic muscle. Quantitative analysis of pulsinginterval vs signal intensity curves (control untreatedmuscle in closed circles, GFP-treated muscle inopen circles, and VEGF165/GFP-treated muscle inopen triangles) demonstrated a greater microvas-cular blood volume, velocity, and overall bloodflow within VEGF165-treated ischemic muscle,compared with other treatment groups.

Figure 3. Noncapillary blood volume(NCBV; A), and capillary blood flow (CBF;B) data in the ischemic muscle, normal-ized to contralateral nonischemic musclefor the 3 treatment groups at 14 days(pretreatment), 28 days and 8 weekspostligation. Increases in blood flow inresponse to VEGF165 treatment comprisedpredominantly of an increase in noncapil-lary blood volume (arteriogenesis), whichwas associated with normalization of CBF.Small increases in NCBV in GFP-plasmidtreated animals did not result in improve-ment in CBF. *P�0.01 compared withcorresponding data in control animals,†P�0.05 compared with correspondingdata in GFP-treated animals, ‡P�0.01compared with corresponding 14 daydata.




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arterioles (50 to 150 �m) (Figure 5B), at this early time pointGFP signal was also detected within capillaries and surround-ing myocytes (Figure 5C).

At day 28, GFP signal was again localized to small tomedium-sized arterioles (Figure 5D). In comparison to the3 day time point, the number of vessels expressing GFP at14 days postdelivery was �5 fold less. In addition, there

was no detectable GFP signal within capillaries or sur-rounding myocytes at this later time point after delivery.By 8 weeks, there was no longer detectable GFP signalwithin treated ischemic muscle. GFP signal was notdetected in the contralateral nonischemic hindlimb, orwithin remote organs, including the lungs, heart, and liver,at any time point.

Figure 4. A, Representative stacked images of microvessels in ischemic hindlimb muscle after no treatment (control), GFP, andVEGF165/GFP therapy at 4 and 8 weeks postligation, using FMA. Compared with normal muscle (inset image in B), control untreatedischemic muscle showed a reduction in microvascular density by FMA that persisted over time. Ultrasound-mediated delivery of GFP-plasmid bearing microbubbles resulted in a mild increase in vessel density at 4 weeks, which did not persist to 8 weeks. After deliveryof VEGF165/GFP plasmid, FMA demonstrated a marked and dense proliferation of neovessels, with an abundance of bridging arterioles.Neovascularization remained present at late time points in VEGF treated muscle. B, Quantitative vessel density by FMA of ischemic andnormal hindlimb muscle. At 28 days postligation, FMA revealed a significant increase in the density of microvessels in the ischemic legof VEGF165-treated animals vs both control and GFP treated groups, with partial regression at week 8. Scale bars 100 �m. *P�0.05compared with normal nonischemic muscle, †P�0.05 compared with corresponding control data, ‡P�0.05 compared with correspond-ing GFP-treated data. (Inset image, FMA of normal hindlimb skeletal muscle).




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Real-time RT-PCR data for exogenous GFP and VEGF165/GFP mRNA is shown in Figure 6. Using specific primers,robust exogenous transgene expression (normalized to thecontralateral nonischemic muscle) was detected in GFP andVEGF165/GFP-treated muscles at day 17 (19.1�12.6 and34.3�25.4, respectively), and was undetectable in controluntreated animals (Figure 6). Both GFP (1.6�0.4) andVEGF165/GFP (1.8�0.6) mRNA expression persisted till day28, but was almost undetectable (1.3�0.6) by 8 weeks inVEGF165/GFP-treated ischemic muscle.

At day 17 endogenous VEGF, Ang-1, and Ang-2 wereupregulated in the ischemic limbs of all groups. Althoughreduced slightly, these endogenous growth factors remainedupregulated in nontreated controls and GFP-treated animalsat day 28 and 8 weeks (Figure 7A). In contrast, in theVEGF165/GFP-treated ischemic muscle at day 28, endogenousVEGF was reduced compared with other treatment groups(Figure 7A). At that time point, endogenous Ang-1 levelswere further increased in the VEGF165/GFP-treated musclecompared with nontreated and GFP-treated ischemic muscle(Figure 7A), and had decreased by 8 weeks postligation.Endogenous VEGF, Ang-1, and Ang-2 mRNA levels innormal muscle from nonligated animals were similar to thoseobtained from the contralateral normal hindlimb muscle inischemic animals (data not shown). Total VEGF proteinlevels by Western blotting followed a similar pattern as both

exogenous and endogenous VEGF mRNA levels, with anearly increase at day 17, a reduction by day 28, and nearnormalization by 8 weeks (Figure 7B).

DiscussionThe potential of ultrasonic destruction of carrier micro-bubbles to deliver genetic material specifically to tissue thatis insonified has been explored over the last decade. Thetherapeutic impact of this strategy has begun to be tested inanimal models of disease. In this study, we have demon-strated that ultrasound-mediated delivery of VEGF165 charge-coupled to microbubbles can improve resting skeletal muscleperfusion in a model of severe peripheral arterial disease.

The ultrasound-facilitated delivery of genes encoding forproangiogenic growth factors (HGF and VEGF) that havebeen combined with microbubbles has recently been demon-strated.12,13 In these studies, improvement in tissue perfusionwas implied by the histologic finding of increased capillarydensity, without assessment of in vivo perfusion. One novelfeature of our current study is that microvascular prolifera-tion, assessed anatomically by fluorescent microangiography,was correlated with recovery of in vivo nutritive perfusion asassessed by CEU. We also confirmed that delivery of geneticmaterial and the biologic effects of the gene product could beaccomplished in chronically and severely hypoperfused mus-cle. The importance of this finding should not be underesti-

Figure 5. Examples of immunofluorescentstaining of a control and VEGF165/GFP-treated animals at various time points afterdelivery. No GFP signal was detected inthe control untreated ischemic muscle (A).At day 17 (3 days postdelivery), a strongand abundant GFP signal (�5-fold greatercompared with day 28 postdelivery),located within the vascular endothelium ofboth arterioles (B) and capillaries (C) wasobserved with GFP signal detected withinmyocytes (arrowhead) adjacent to capillarynetworks (C). By day 28, GFP signal wasdetected predominantly within the vascu-lar endothelium of small to medium sizedarterioles (D).




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mated because microbubble transit through ischemic skeletalmuscle is expected to be quite low. Previous studies exam-ined delivery of proangiogenic genes to tissue adjacent12 toor directly within13 normal myocardium which has a flowrate at least 20-fold greater than that in ischemic skeletal

muscle at rest. Another unique feature of this study was thefinding that exogenous VEGF165 treatment suppressedendogenous rat VEGF mRNA levels, suggesting that reso-lution of ischemia also reversed native angiogenic growthfactor upregulation.19,20

Figure 6. RT-PCR data showing exogenous GFP and VEGF165/GFP mRNA transcript at day 17, day 28, and 8 weeks. GFP and VEGF/GFP165 mRNA expression at day 17 was high, and was significantly greater than mRNA expression at day 14 postdelivery. Exogenoustransgene expression remained detectable at low levels at day 14 and was almost undetectable at 8 weeks. *P�0.001 compared withcorresponding day 28 data, †P�0.05 compared with corresponding control data.

Figure 7. A, RT-PCR for endogenous gene expression, VEGF (solid bars), Ang-1 (open bars), and Ang-2 (hatched bars). Growth factors(normalized to the contralateral non-ischemic leg) were upregulated at day 17 in all groups, being more pronounced in the VEGF/GFP-treated animals. At day 28, there was a significant downregulation of VEGF and upregulation of Ang-1 in the VEGF165/GFP-treatedgroup compared with both GFP and untreated control ischemic muscle, suggesting modulation of the endogenous angiogenicresponse. By 8 weeks, endogenous growth factor gene expression returned to near baseline levels in all groups. *P�0.05 comparedwith corresponding data in control and GFP-treated muscle. B, Total VEGF protein levels by Western blotting in VEGF/GFP-treatedanimals at day 17, day 28, and week 8. Corresponding to exogenous VEGF/GFP and endogenous VEGF mRNA levels, there was anincrease in total VEGF at day 17, with a reduction at day 28, and a return to near baseline levels at week 8. *P�0.05 compared withnormal hindlimb muscle.




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In our study, transfection of our targeted genes wasconfirmed by the detection of GFP signal within tissue byfluorescent confocal microscopy and by the presence ofexogenous mRNA expression on real-time RT-PCR. Theformer technique allowed us to localize gene transfectionprimarily to the endothelial layer of vessels. Early afterdelivery, both arterioles and capillaries expressed GFP, witha greater proportion of vessels targeted being arterioles. Atearly time intervals, GFP signal was also seen in myocytesadjacent to GFP-positive capillaries. At day 14 postdelivery,GFP was localized only to arterioles. These findings areconsistent with previous studies showing localization offluorescent plasmid mainly to the vascular and perivascularregions after ultrasound-mediated delivery.10 Thus, the ultra-sonic destruction of intravascular carrier microbubbles targetsthe vascular endothelium, making this technique ideallysuited to gene delivery for therapeutic angiogenesis. Thelatter method, real-time RT-PCR, was used to confirm theproduction of exogenous mRNA by our transfected genes,using an ultra-sensitive technique. The timing of mRNAexpression in our study is in keeping with the findings ofBekeredjian et al11 where transgene expression peaked withinthe first 4 days, with a rapid decline thereafter. We did notfind increases in MBF at day 3 after delivery, suggesting thatthe neovascularization response to gene delivery followedpeak mRNA expression. Although vascular density and MBFpartially regressed very late after gene delivery, they re-mained increased as compared with baseline pretreatmentvalues, consistent with a persistence of neovascularization ata time point when therapeutic transgene expression hadceased. This would imply that repeated doses delivered overtime will likely be required to produce a sustained angiogenicresponse, thus further emphasizing the importance of anoninvasive method of gene delivery.

Another novel feature of this study was the application ofCEU to differentiate the functional expansion of the capillaryand noncapillary microvascular compartments. These dataindicated that the improvement of capillary perfusion wasassociated with an increase in noncapillary microvascularblood volume. It was impossible to confirm that arteriolardelivery of VEGF plasmid has a predominant effect on thesame vessels. However, our findings are in agreement withthe idea that remodeling of the resistance arteriolar bed is themajor determining factor for reducing network resistance inthe presence of a proximal stenosis and recovery of tissueperfusion.21 Ultrasound-mediated delivery of GFP-plasmidbearing microbubbles also increased noncapillary blood vol-ume, or arteriogenesis, although to a lower degree. Previousstudies have demonstrated that the biologic effects ofultrasound-mediated microbubble destruction results in thepromotion of arteriogenesis in rat skeletal muscle.22,23 Postu-lated mechanisms for this biologic effect have included therecruitment of inflammatory cells, platelets, or bone marrowstem cells,23 the release of platelet-derived proinflammatoryfactors which may attract circulating endothelial progenitorcells,24 or the via the recruitment of VEGF-producing inflam-matory cells.25 Unlike these studies, we found that controlmicrobubble destruction did not result in increases in total orcapillary blood flow, and are in keeping with other studies of

ultrasound-mediated gene delivery.12,13 Differences in ultra-sound transmit frequency and acoustic power, microbubblecomposition and doses, methods of blood flow determination,and vessel density measurements between studies could haveaccounted for these differences. With the doses and methodsin our study, however, control microbubble destruction didnot alter endogenous growth factor expression, and thechanges in noncapillary blood volume were not sufficientenough to improve nutritive capillary blood flow. Regardless,we demonstrated that the use of plasmid encoding the growthfactor VEGF, as compared with one encoding only a markerprotein GFP, resulted in a substantially greater “angiogenic”effect.

RT-PCR data on endogenous growth factor mRNA expres-sion offers unique insights into the biology of angiogenesis.VEGF is consistently elevated early after ischemia in theheart20,26 and skeletal muscle,19 and likely plays a role early inthe endogenous angiogenic response to ischemia.20,26 There isincreasing evidence that angiopoietins, Ang-1 and Ang-2, arealso important for blood vessel formation.27,28 Ang-2 plays asynergistic role with VEGF early in the angiogenic response,with both being upregulated early after ligation in our study.In contradistinction, Ang-1 appears to be play an importantrole relatively later in the angiogenic process by contributingto stabilization and maturation of neovessels29,30 and oppos-ing the actions of VEGF.31 In our study, endogenous VEGFand Ang-2 mRNA levels were reduced and Ang-1 mRNAlevels further upregulated at day 28 in the group receivingVEGF165-bearing microbubbles, as compared with all othertreatment groups. This finding is consistent with a reversal ofearly VEGF/Ang-2 upregulation with exogenous VEGF165

therapy that was not seen in other treatment groups. Westernblotting for total VEGF protein levels confirmed an earlyelevation 3 days after VEGF delivery, and a later reduction atday 28, when exogenous transgene expression had waned.Importantly, the upregulation in Ang-1 at day 28 is consistentwith the process of neovessel maturation, a late event in theangiogenic response. Taken together, these observations pro-vide further compelling evidence for a biologic effect19,20 ofour ultrasound-mediated gene delivery.

There are several limitations to our present study. We onlyexamined a single large dose of VEGF165 at a single timepoint, which was highly effective. Defining the dose-responserelationship for this delivery technique remains important.Given the number and complexity of the factors determiningthe efficacy and longevity of UM gene delivery, including (1)plasmid DNA and cationic microbubble doses and concen-trations, (2) ultrasound acoustic power and pulsing interval,and (3) the effects of repeated deliveries over time, this isbeyond the scope of our present study, however remains thegoal of future studies. Although we believe that our findingscan potentially translate into an effective noninvasive methodof therapeutic gene delivery in humans, our present studyonly provides proof of principle. Many potential obstaclesexist, including (1) determining the most safe and effectiveultrasound settings and microbubble concentrations for hu-man use, given concerns over the use of high acousticpowers, (2) defining a dose-response relationship in largeanimal models, in applicable organs, such as the heart, that




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more closely mimics the clinical conditions, and (3) thedevelopment of ultrasound probes, designed specifically forhuman delivery.

In conclusion, ultrasound-mediated microbubble destruc-tion using VEGF165 plasmid-bearing microbubbles results intargeted transfection of the vascular endothelium, leading toarteriogenesis and improved tissue perfusion in the setting ofsevere chronic hypoperfusion. This noninvasive techniqueholds great promise as a method to target angiogenic genetherapy to ischemic tissue, in any organ accessible toultrasound.

Sources of FundingThis work was supported by an Operating Grant from the CanadianInstitutes of Health Research, Ottawa, Ontario, Canada, and anEquipment Grant from the Canadian Foundation for Innovation,Ottawa, Ontario, Canada. Dr Leong-Poi is supported by a NewInvestigator Award from the Canadian Institutes of Health Research,Ottawa, Ontario, Canada. Dr Lindner is supported by grants R01-HL-074443, R01-HL-078610, and R01-DK-063508 from the Na-tional Institutes of Health, Bethesda, Md.

DisclosuresNone.

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Teichert-Kuliszewska, Alexander L. Klibanov, Duncan J. Stewart and Jonathan R. LindnerHoward Leong-Poi, Michael A. Kuliszewski, Michael Lekas, Matthew Sibbald, Krystyna

Chronically Ischemic Skeletal Muscle Plasmid Gene Delivery to165Therapeutic Arteriogenesis by Ultrasound-Mediated VEGF

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2007 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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Online Supplement Leong-Poi et al. Therapeutic Arteriogenesis by Ultrasound-Mediated…

EXPANDED MATERIALS AND METHODS

Microbubble and DNA Preparation

Plasmid DNA was charge-coupled to cationic lipid microbubbles as previously

described.1 Microbubbles with a cationic (zeta potential of +60 mV) lipid shell2 were

created, which when incubated with plasmid DNA, results in approximately 6700

plasmids on surface of each microbubble.1 For perfusion imaging, non-targeted lipid-

shelled decafluorobutane microbubbles (MP1950) were used. Microbubble

concentrations were determined by electrozone sensing with a Coulter Multisizer IIe

(Beckman-Coulter).

Plasmid vectors were constructed for transfection of enhanced green fluorescent

protein (GFP) alone or the co-transfection of both human VEGF165 and GFP. For the

latter, we constructed a bicistronic vector encoding human VEGF165 and GFP, which

incorporates an internal ribosome entry site (IRES) that facilitates the translation of both

proteins from a single mRNA molecule, with high levels of expression of both genes

within the same cell.3

Animal Preparation

The study protocol was approved by the Animal Care and Use Committee at St.

Michael’s Hospital-Health Sciences Research Centre, University of Toronto. Proximal

hindlimb ischemia was produced in 90 Sprague-Dawley rats. Rats were anesthetized with

intraperitoneal injection of ketamine hydrochloride (10 mg·kg-1) and xylazine (8 mg·kg-

1). Using aseptic technique, the left common iliac artery and small proximal branches


were exposed and ligated with 4-0 suture. The incision was closed in layers and animals

were recovered.

Perfusion Imaging

Contrast-enhanced ultrasound (CEU) imaging of the proximal hindlimb adductor

muscles was performed with gated pulse inversion imaging (HDI 5000, Philips

Ultrasound) at a mechanical index of 1.0 and a transmission frequency of 3.3 Mhz (L7-4

transducer). Gain settings were optimized and held constant. Data were recorded on

magnetic-optical disk, and transferred to a computer for off-line analysis.

Perfusion in the adductor muscles was assessed during continuous i.v. infusion of

non-targeted MP1950 microbubbles (1×107 min-1). Background images were acquired at

baseline for subtraction of tissue signal. Intermittent imaging was then performed by

progressive prolongation of the pulsing interval (PI) from 0.2 to 20 s, using the internal

timer. Several averaged background frames were digitally subtracted from averaged

contrast-enhanced frames at each PI. PI versus signal intensity (SI) data were fit to the

function, y = A (1-e-βt), where y is SI at the pulsing interval t, A is plateau video

intensity which is an index of microvascular blood volume (MBV), and β is the rate

constant which provides a measure of microvascular blood velocity.4 Microvascular

blood flow (MBF) was calculated by the product of A and β.

CEU perfusion data were recalculated using averaged frames at a PI of 1 s as

background to eliminate signal from vessels with a transaxial plane velocity of 2.4×10-3

m/s.5 This process eliminates signal from almost all non-capillary microvessels, with

minimal loss of capillary signal, thereby yielding information from the capillary

compartment alone. The relative non-capillary blood volume (NCBV) was determined by


the difference of the calculated A-values. This algorithm has been previously used to

measure dynamic changes in skeletal muscle capillary volume, and has been validated

against assays for capillary xanthine oxidase availability.5

Ultrasound-Targeted Gene Delivery

For gene delivery, ultrasound transmission was performed with a phased array

transducer (Sonos 5500, Philips Ultrasound) at 1.3 MHz using B-mode ultraharmonic

imaging at a transmit power of 0.9W (120 V, 9 mA). Cationic microbubbles (1x109)

coupled with 500 µg of cDNA were infused intravenously over 10 minutes. To allow for

a wider field of delivery, the transducer was positioned transverse to the ischemic

adductor muscles and ultrasound was transmitted during a slow sweep along the length of

the proximal hindlimb muscles. A PI of 5 s was used to allow microbubble replenishment

into the beam elevation between each pulse of ultrasound. Ultrasound transmission was

continued for 10 minutes after cessation of the infusion, to destroy remaining circulating

DNA-loaded microbubbles.

Fluorescent Microangiogaphy (FMA)

Prior to sacrifice, the abdominal aorta was cannulated and the distal hindlimbs

flushed with 20 mL of heparinized saline. A 10% solution of fluorescent microspheres

(2µm diameter) (Sigma) mixed with a 1% solution of low melting point agarose at 45oC

was slowly injected into the aortic cannula. The animal was euthanized and placed in an

ice bath to facilitate rapid cooling and solidification of the casting agent. Hindlimb

muscle was removed and placed in 10% buffered formalin and sectioned (200 µm).

Using confocal microscopy, a series of stacked images (4µm slices) was taken and the

middle 25 slices (100 µm total thickness) were projected in order to quantify the density


of blood vessels using automated software (IPTK analysis software, Reindeer Graphics

Inc.). This technique has been previously described to quantify pulmonary vascular

density in experimental models of pulmonary arterial hypertension.6

Immunohistochemistry

In vivo transfection efficacy and spatial localization was determined using

immunohistochemistry. Explanted tissue was cryo-embedded in OCT (Sakura, Japan)

and stored at -80oC. The cryo-blocks were sectioned (15 µm thick) every 25 µm and re-

hydrated in phosphate buffered saline (PBS) for 30 min, fixed in 2% paraformaldehyde

(PFA) (Sigma) in PBS for 10 minutes, and washed 3 times with PBS. Cell surface

antigens were identified using: mouse anti-human CD31 (Alpha Diagnostics Intl., Inc.),

mouse anti-human Tie-2 (Clone Ab33, Upstate Biotechnology), UEA-1 (Sigma) and

mouse anti-human Alpha-actin (Sigma). The presence of antibody was confirmed by

exposure to a phycoerythrin (PE) conjugated secondary antibody. TOPO-3 (Sigma) was

used as a nuclear marker.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Semi-quantitative real-time RT-PCR for endogenous rat VEGF, angiopoietin-1

(Ang-1) and Angiopoietin-2 (Ang-2) mRNA, as well as exogenous GFP and

VEGF165/GFP transcripts were performed using standard techniques in our laboratory.

Tissue was homogenized using Trizol (Sigma), after which total RNA was isolated using

the GenElute Mammalian total RNA kit (Sigma) and quantified by absorbance at 260 nm.

Total RNA was reverse transcribed in 20 µl volumes using Omniscript RT kit (Qiagen)

with 1 µg of random primers. For each RT product, aliquots (2-10 µl) of the final

reaction volume were amplified by real-time PCR reactions using standardized


concentration of RNA. Rat VEGF, Ang-1, Ang-2, GFP, VEGF165/GFP and cyclophilin

specific primers and SYBR green (Applied BioSystems) were then used to detect

amplicon production using an ABI system.

Western blotting

Western blotting was performed to measure total VEGF165 protein levels in

ischemic hindlimb muscle at various time points post ligation in the VEGF-treated group,

using standard techniques in our laboratory. Frozen tissue samples were homogenized in

RIPA lysis buffer. Total protein (100 µg/line) was subjected to SDS-polyacrylamide gel

electrophoresis (PAGE) in 10-12% Tris-glycine gels (Invitrogen) under non-reduced

condition, subsequently transferred to nitrocellulose membranes and blocked with 5%

BSA in TBST buffer (10mmol/L Tris 150mmol/L NaCl pH 7.5 and 0.1% Tween 20). The

membranes were probed overnight with goat anti-human antibodies to VEGF165 (R&D)

at concentration 0.2µg/ml in TBST with 3% BSA, followed by an anti-goat IgG

secondary antibody conjugated to horseradish peroxidase (1:3000, 1hr; Promega). After

being stripped, the membranes were reprobed with monoclonal antibody against

Glyceraldehyde-3-Phosphatase Dehydrogenase (GAPDH) from skeletal muscle (1:5000,

2hr; Chemicon, Millipore) for the estimation of total protein loaded. Specific bands were

visualized using an enhanced chemiluminescence substrate system ECL (Amersham

Pharmacia Biotech). Densitometry was performed and the intensity of each band was

analyzed using the Molecular Analyst software (Imaging Densitometer, Bio-Rad).

Experimental Protocol

CEU perfusion imaging of both hindlimb adductor muscles was performed 14

days after iliac artery ligation. Ultrasound-targeted gene delivery was then performed,


according to assigned treatment group: group 1- control, no treatment; group 2- GFP

plasmid; group 3- VEGF165/GFP plasmid (n=18 per group). Repeat CEU was performed

at days 17 (n=6 per group) and 28 (n=12 per group). In order to assess late gene

transfection and efficacy, an additional 36 rats (n=12 per treatment group), were studied

at 8 weeks post-ligation. In 4 rats per group, FMA was performed immediately prior to

sacrifice. In remaining animals, skeletal muscle tissue from the ischemic and non-

ischemic adductor muscles, as well as tissue from the lungs, heart and liver was obtained

for post-mortem immunohistochemistry, quantitative RT-PCR and Western blotting.

Normal hindlimb muscles from an additional 6 animals without ligation were obtained for

semi-quantitative RT-PCR, for comparison to hindlimb muscles from ischemic animals

that underwent ligation.

Statistical Methods

Data are expressed as mean±SD. Comparisons between multiple stages were

made with one-way ANOVA. When differences were found, inter-stage comparisons

were performed using non-paired Student’s t-test with Bonferroni correction. Data for

pre- and post-gene therapy were compared by paired Student’s t-test. Differences were

considered significant at p <0.05 (2-sided).


Reference List

(1) Christiansen JP, French BA, Klibanov AL, Kaul S, Lindner JR. Targeted tissue

transfection with ultrasound destruction of plasmid-bearing cationic

microbubbles. Ultrasound Med Biol. 2003;29:1759-1767.

(2) Unger EC, McCreery TP, Sweitzer RH. Ultrasound enhances gene expression of

liposomal transfection. Invest Radiol. 1997;32:723-727.

(3) Ye L, Haider HK, Jiang S, Ge R, Law PK, Sim EK. In Vitro Functional

Assessment of Human Skeletal Myoblasts After Transduction With Adenoviral

Bicistronic Vector Carrying Human VEGF(165) and Angiopoietin-1. J Heart

Lung Transplant. 2005;24:1393-1402.

(4) Wei K, Jayaweera AR, Firoozan S, Linka A, Skyba DM, Kaul S. Quantification

of myocardial blood flow with ultrasound-induced destruction of microbubbles

administered as a constant venous infusion. Circulation. 1998;97:473-483.

(5) Dawson D, Vincent MA, Barrett EJ, Kaul S, Clark A, Leong-Poi H, Lindner JR.

Vascular recruitment in skeletal muscle during exercise and hyperinsulinemia

assessed by contrast ultrasound. Am J Physiol Endocrinol Metab. 2002;282:E714-

E720.

(6) Zhao YD, Courtman DW, Deng Y, Kugathasan L, Zhang Q, Stewart DJ. Rescue

of monocrotaline-induced pulmonary arterial hypertension using bone marrow-

derived endothelial-like progenitor cells: efficacy of combined cell and eNOS

gene therapy in established disease. Circ Res. 2005;96:442-450.

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