The FASEB Journal • Research Communication

Therapeutic angiogenesis due to balanced single-vectordelivery of VEGF and PDGF-BB

Andrea Banfi,*,†,‡,1 Georges von Degenfeld,*,2 Roberto Gianni-Barrera,†,‡

Silvia Reginato,†,‡ Milton J. Merchant,* Donald M. McDonald,§,�,¶ and Helen M. Blau*,1

*Baxter Laboratory for Stem Cell Biology, Institute for Regenerative Medicine and Stem CellBiology, Department of Microbiology and Immunology, Stanford University, Stanford, California, USA;†Department of Biomedicine and ‡Department of Surgery, Basel University Hospital, Basel,Switzerland; and §Cardiovascular Research Institute, �Comprehensive Cancer Center, and¶Department of Anatomy, University of California at San Francisco, San Francisco, California, USA

ABSTRACT Therapeutic angiogenesis by delivery ofvascular growth factors is an attractive strategy fortreating debilitating occlusive vascular diseases, yetclinical trials have thus far failed to show efficacy. As aresult, limb amputation remains a common outcomefor muscle ischemia due to severe atherosclerotic dis-ease, with an overall incidence of 100 per millionpeople in the United States per year. A challenge hasbeen that the angiogenic master regulator vascularendothelial growth factor (VEGF) induces dysfunc-tional vessels, if expressed outside of a narrow dosagewindow. We tested the hypothesis that codelivery ofplatelet-derived growth factor-BB (PDGF-BB), whichrecruits pericytes, could induce normal angiogenesis inskeletal muscle irrespective of VEGF levels. Coexpres-sion of VEGF and PDGF-BB encoded by separatevectors in different cells or in the same cells onlypartially corrected aberrant angiogenesis. In markedcontrast, coexpression of both factors in every cell at afixed relative level via a single bicistronic vector led torobust, uniformly normal angiogenesis, even whenVEGF expression was high and heterogeneous. Nota-bly, in an ischemic hindlimb model, single-vector ex-pression led to efficient growth of collateral arteries,revascularization, increased blood flow, and reducedtissue damage. Furthermore, these results were con-firmed in a clinically applicable gene therapy approachby adenoviral-mediated delivery of the bicistronic vec-tor. We conclude that coordinated expression of VEGFand PDGF-BB via a single vector constitutes a novelstrategy for harnessing the potency of VEGF to inducesafe and efficacious angiogenesis.—Banfi, A., von De-genfeld, G., Gianni-Barrera, R., Reginato, S., Mer-chant, M. J., McDonald, D. M., Blau, H. M. Therapeutic

angiogenesis due to balanced single-vector delivery ofVEGF and PDGF-BB. FASEB J. 26, 2486–2497 (2012).www.fasebj.org

Key Words: ischemia � gene therapy � adenoviral vectors

Atherosclerotic coronary artery disease and pe-ripheral vascular disease remain major causes of mor-bidity and mortality, despite medical and surgical ad-vances, with a prevalence of 15–20% of all people �70yr of age in the U.S. (1). Therapeutic angiogenesis, thegrowth of new blood vessels promoted by delivery ofvascular growth factors, is well accepted as a strategythat could fill this currently unmet medical need, but todate efforts to achieve this goal have largely failed.Challenges include the need to combine methods forefficient expansion of the microvasculature of ischemictissue with enlargement of upstream collateral arteries(arteriogenesis) in order to restore blood flow tohypoxic muscle tissue.

Vectors that overexpress vascular endothelial growthfactor (VEGF), the master regulator of angiogenesis,can drive robust angiogenesis, but the remarkablynarrow dosage range of VEGF expression compatiblewith normal vessel formation has hindered its effica-cious use (2, 3). VEGF binds tightly to the extracellularmatrix (4) and induces normal or aberrant vasculargrowth depending on its localized dosage in the mi-croenvironment (3, 5). Consequently, VEGF has thepotential to induce significant adverse effects, such asincreased blood vessel permeability with edema (6),and aberrant vascular proliferation with angioma-like

1 Correspondence: H.M.B., Baxter Laboratory for Stem CellBiology, Stanford University School of Medicine, CCSR 4216,269 Campus Dr., Stanford, CA 34305-5175, USA. E-mail:hblau@stanford.edu; A.B., Cell and Gene Therapy, BaselUniversity Hospital, ICFS 407, Hebelstrasse 20, Basel, CH-4031 Switzerland. E-mail: abanfi@uhbs.ch

2 Current address: Common Mechanism Research, BayerHealthcare AG, D-42096 Wuppertal; Department of Experi-mental Cardiology, University of Witten-Herdecke, Germany

doi: 10.1096/fj.11-197400

Abbreviations: �SMA, �-smooth muscle actin; H&E, hema-toxylin and eosin; IRES, internal ribosomal entry site; LacZ,�-galactosidase; NG2, nerve/glial antigen 2; P cell, PDGF-BB-expressing cell; PDGF-BB, platelet-derived growth factor-BB;SCID, severe combined immunodeficiency; sPDGFR-�, solu-ble platelet-derived growth factor receptor-�; V cell, VEGF-expressing cell; VEGF, vascular endothelial growth factor; VIPcell, VEGF–IRES–PDGF-BB–expressing cell; VLD, vessellength density

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growth (7) if even rare “hotspots” of excessive expres-sion occur (3).

Expansion of the microvascular bed following VEGFdelivery has been shown to lead to arteriogenesis byincreasing blood flow and shear stress (8) and inducingupstream responses through retrograde conductionalong vessel walls via intercellular gap junctions (9).However, controlled clinical trials employing a varietyof vectors and growth factors have failed to show clearefficacy (10). Indeed, retrospective analyses have shownthat when VEGF was delivered at a safe dose, it did notgenerate sufficient angiogenesis to correct cardiac orskeletal muscle ischemia (11, 12). Therefore, the cur-rent conundrum is that increased VEGF levels arerequired to achieve beneficial effects, yet high expres-sion levels readily lead to a loss of safety.

Why have the approaches used to date failed? Growthof normal blood vessels requires the complex interac-tion of multiple cell types and growth factors that arecoordinated in time and space (13). We postulated thata “well-tempered vessel”—like Bach’s “well-temperedclavier”—could be achieved by balancing the effects ofexogenously delivered VEGF with endogenous factors(14). Blood vessel stability depends on the coordinatedinteraction of multiple signaling pathways in the endo-thelium and pericytes (13). VEGF promotes endothe-lial cell growth, while platelet-derived growth factor-BB(PDGF-BB) stabilizes blood vessels by recruiting peri-cytes (13). When blood vessel growth is induced byother proangiogenic factors, such as placental growthfactor, fibroblast growth factor-2, and hepatocytegrowth factor, VEGF is up-regulated (15). Accordingly,therapeutic strategies that deliver a single angiogenicagent must inevitably depend on the availability ofendogenous factors to achieve a balance. Without thatbalance, the new blood vessels are abnormal, unstable,and inefficient (2, 3, 14, 16, 17). Delivery of VEGFalone can achieve such a balance, but only if itsexpression is tightly controlled in every transduced cellwithin a specific range of levels (2, 3, 5), and thiscontrol has yet to be achieved with current genetherapy approaches.

Here we test the hypothesis that a combination offactors that promote both endothelial cell proliferationand pericyte recruitment, VEGF and PDGF-BB, candrive the growth of normal blood vessels and reversemuscle ischemia. Notably, we focused on angiogenesisin limb skeletal muscle, because this is the clinicallyrelevant tissue in which ischemia occurs (12). Indeed,the importance of this choice of target tissue is high-lighted by the disparate effects of angiogenic growthfactors seen by others in nonmuscle tissue microenvi-ronments in which the local factor milieu likely differs(18, 19). Our data suggest that a bicistronic vectorencoding VEGF and PDGF-BB in a fixed balanced ratiowill constitute an efficacious viral vector-mediated genetherapy strategy for the treatment of debilitating mus-cle ischemia associated with atherosclerotic vasculardisease.

MATERIALS AND METHODS

Cell culture

C57BL/6 mouse primary myoblasts, already transduced toexpress the �-galactosidase marker gene from a retroviralpromoter (20), were further infected at high efficiency, asdescribed previously (7), with retroviruses expressing murineVEGF164, or human PDGFb, or both. Early-passage myoblastclones were randomly isolated using a FacStar cell sorter(Becton Dickinson, San Jose, CA, USA) as described previ-ously (3), and single-cell isolation was confirmed visually. Allmyoblast populations were cultured in 5% CO2 on collagen-coated dishes as described previously (20).

VEGF and PDGF-BB ELISA measurements

The amounts of mVEGF164 and hPDGF-BB in cell culturesupernatants and whole-muscle tissue lysates were quantifiedusing specific ELISA kits (R&D Systems, Abingdon, UK) asdescribed previously (3). Briefly, cell culture supernatants(n�4) were harvested after 4 h incubation with fresh mediumsupplemented with 10 �g/ml heparin to prevent retention ofPDGF-BB on the cell surface. Results for VEGF and PDGF-BBare expressed relative to the number of cells and time ofincubation (ng/106 cells/d). The results for tissue lysateswere normalized for the total amount of protein, quantifiedwith the BC protein assay (Bio-Rad, Reinach, Switzerland).

In vivo myoblast implantation

Severe combined immunodeficiency (SCID) CB.17 mice(6–8 wk old; Taconic, Germantown, NY, USA) were treatedin accordance with the U.S. National Institutes of Health andSwiss federal guidelines for animal welfare, with protocolsapproved by the Stanford University Administrative Panel onLaboratory Animal Care and the Veterinary Office of theCanton Basel-Stadt. Myoblasts (5�105) in 5 �l of PBS with0.5% BSA were injected into the tibialis anterior (calf) or theauricularis posterior (dorsal aspect of the external ear) mus-cles using a 29½-gauge needle.

Recombinant adenovirus production

Recombinant adenoviruses expressing either mouse VEGF164,human PDGF-BB, or both in a bicistronic cassette were pro-duced using the Adeno-X Expression System (Clontech, Saint-Germain-en-Laye, France) according to manufacturer’s recom-mendations. All adenoviral constructs also expressed a truncatedversion of CD8 as a marker gene. Briefly, target genes werecloned into the pShuttle vector, subcloned into the Adeno-Xviral DNA, and used to transfect HEK293 cells with Fugene HDreagent (Roche Applied Science, Basel, Switzerland). After 1 wk,viral particles were collected from transfected cells by repeatedfreezing–thawing and used for reinfection of fresh HEK293cells. After 4–5 lysis and infection cycles, viral particles werecollected and purified by a double cesium chloride gradient.Viral titer was determined as infectious units after serial infec-tion of HEK293 cells at different multiplicities of infection, asdescribed previously (21). Adenoviral vectors were diluted inphysiological solution and injected in ear and hindlimb musclesof SCID mice at the titer of 1 � 108 infectious units/injection.

Tissue staining

The entire vascular network of the ear was visualized byintravascular staining with a biotinylated Lycopersicon esculen-

2487BALANCED VEGF AND PDGF-BB FOR NORMAL ANGIOGENESIS

tum lectin (Vector Laboratories, Burlingame, CA, USA) thatbinds the luminal surface of all blood vessels, as describedpreviously (3). Mice were anesthetized, lectin was injectedintravenously, and 2 min later, tissues were fixed by vascularperfusion of 1% paraformaldehyde and 0.5% glutaraldehydein PBS (pH 7.4). Ears were removed, bisected in the plane ofthe cartilage, and stained with X-gal to detect implantedmyoblasts. Vessels were stained with avidin-biotin complex-diaminobenzidine histochemistry (Vector Laboratories, Bur-lingame, CA, USA), dehydrated through an alcohol series,cleared with toluene, and whole-mounted on glass slides withPermount embedding medium (Fisher Scientific, Wohlen,Switzerland).

For tissue sections, mice were sacrificed by cervical dislo-cation. Tibialis anterior muscles were harvested and frozen inoptimal cutting temperature (OCT) compound (SakuraFinetek, Torrance, CA, USA). Sections were stained withX-gal or with hematoxylin and eosin (H&E). Immunostainingwas performed using the following antibodies and dilutions:rat monoclonal anti-mouse CD31 (BD Biosciences, Basel,Switzerland; 1:100); mouse monoclonal anti-mouse �-smoothmuscle actin (�SMA; MP Biomedicals, Basel, Switzerland;1:400); rabbit polyclonal anti-nerve/glial antigen 2 (NG2;Chemicon International, Chandlers Ford, UK; 1:200);chicken polyclonal anti-laminin (Abcam, Cambridge, UK;1:200). Fluorescently labeled secondary antibodies (Invitro-gen, Basel, Switzerland) were used at 1:100. As areas of effectdid not extend to the whole muscle but were limited to theimplantation sites, areas of engraftment were unequivocallyidentified by tracking implanted myoblasts by X-gal stainingin serial parallel sections. Images were acquired with anOlympus BX61 epifluorescence microscope (Olympus, To-kyo, Japan) and AnalySIS D acquisition software (Soft Imag-ing System, Münster, Germany) at the lowest magnificationthat could adequately reveal the structure of the inducedvessels. Composite fluorescent images were generated byoverlaying the different channels with Adobe Photoshop CS2(Adobe Systems, San Jose, CA, USA).

Vessel measurements

Vessel length density (VLD) and diameters were measured inwhole mounts of lectin-stained ears as described (3). Vesselswere randomly chosen by overlaying microscopic images witha computer-generated grid, and 200–300 total vessel diame-ters were measured from 4–5 ears/group. VLD was measuredon 3–6 fields/ear and 4–5 ears/group by tracing the totallength of vessels with AnalySIS D software (Soft ImagingSystem) and dividing it by the area of the fields. In ischemiaexperiments VLD was measured on cryosections of the distalthigh muscles (quadriceps femoris and adductor muscles),immunostained for CD31, on 3 randomly acquired imagesper group. Centerlines of vessels were manually drawn asdescribed previously (3), quantified using calibrated Openlabimage analysis software (Improvision, Lexington, MA, USA)and normalized to the number of muscle fibers.

Plasma leakage measurements

Evans blue assays were performed as described previously (6).Briefly, Evans blue dye (30 mg/kg in 100 �l PBS; J. T. Baker,Phillipsburg, NJ, USA) was injected i.v. After 4 h, mice wereperfused with 1% paraformaldehyde in 0.05 M citric acid (pH3.5). Biopsy punches (6 mm) of ears were obtained (SklarInstruments, West Chester, PA, USA). Evans blue was ex-tracted from tissue with formamide at 55°C overnight andmeasured with a spectrophotometer at 610 nm. Plasmaleakage was measured at 4, 7, and 14 d after myoblast

implantation and expressed as nanograms of dye per milli-gram of tissue wet weight (n�5). Total leakage was alsonormalized to the total vascular surface induced by thedifferent myoblast populations in similarly injected ears(n�4–5 ears/condition). Total vascular surface was estimatedas the product of average vessel perimeter (� � averagediameter, measured as described above) and total vessellength (VLD, measured as described above, multiplied by thetotal area of effect measured on low-magnification micro-scopic pictures). Normalized leakage was expressed as nano-grams of dye per square millimeter of vascular surface in thearea of effect (n�5).

Hindlimb ischemia

Male SCID CB.17 mice (16–20 wk old) were treated accord-ing to the guidelines of the Stanford University Administra-tive Panel on Laboratory Animal Care. Mice were maintainedunder isoflurane anesthesia. Unilateral hindlimb ischemiawas induced by ligation and transection of the medial portionof the right superficial femoral artery distal to the deepfemoral artery origin as described previously (5). Mice wererandomized to receive either vehicle or 8 � 106 total myo-blasts suspended in 0.5% BSA in PBS, as 8 injections into thedistal thigh muscles (5–7 mice/group).

After 14 d, blood flow was measured using fluorescentmicrospheres (5). Red fluorescent microspheres (2�105, 15�m diameter; Invitrogen) were continuously injected over 60s into the beating left ventricle. The heart was perfused withTris-HCl buffer containing Na�, Ca2�, Mg2�, and 0.1%adenosine (2 min), followed by 1.5% formaldehyde (2 min).The thigh muscle group (adductor and quadriceps) wasexcised, cut in midthigh, weighed, and snap-frozen in OCTcompound. Microspheres were individually counted by directfluorescence microscopy on 100-�m cryosections from theentire distal thigh muscles, where the different myoblastpopulations had also been implanted. These sections wereserial to those used for histological analysis of vascular mor-phology. Microsphere counts were normalized by muscleweight, and the counts from the ischemic leg were expressedas a percentage of those in the contralateral, nonischemic leg.Kidneys were analyzed as reference organs to confirm equiv-alent bilateral microsphere distribution.

Collateral vessels �30 �m in diameter were quantified oncross sections of the proximal adductor muscle by costainingfor CD31 and �SMA (5). Damaged muscle was identified onH&E-stained cryosections of the calf muscles as either inflam-mation (mononucleated cell infiltrates) or necrosis (“ghostfibers” lacking nuclei). Areas were manually drawn on digitalimages and quantified using calibrated Openlab image anal-ysis software.

Protein isolation and quantification

Whole fresh mouse muscles were disrupted using a QiagenTissue Lyser (Qiagen, Hombrechtikon, Switzerland) in 500 �lof PBS � 1% Triton X-100, supplemented with CompleteProtease Inhibitor Cocktail (Roche Diagnostics, Rotkreuz,Switzerland), which does not denature proteins or lyse nuclei.After centrifugation, 200-�l aliquots of the lysates were usedfor protein quantification and ELISA analysis.

Statistics

Data are presented as means se. The significance ofdifferences was evaluated using analysis of variance (ANOVA)followed by the Bonferroni test. Values of P 0.05 wereconsidered statistically significant.

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RESULTS

Uncoordinated VEGF and PDGF-BB codeliveryresults in both normal and aberrant angiogenesis

We first investigated the effects of expressing eitherVEGF or PDGF-BB alone on angiogenesis. For thispurpose, we employed a previously described modelsystem entailing primary mouse myoblast-based genedelivery (20), which allows expression of genes in aspatially and temporally controlled manner. �-Galacto-sidase-expressing cells were further transduced to pro-duce either an isoform of VEGF-A (VEGF164, desig-nated here as VEGF) or PDGF-BB linked to a truncatedcell surface marker CD8a, whereas control cells weretransduced with CD8a only (V, P, and CD8, respec-tively, Fig. 1A). In vitro, V cells produced 112 10.4ng/106 cells/d of VEGF, and P cells secreted 45.7 2.4ng/106 cells/d of PDGF-BB. Genetically engineeredmyoblasts were implanted into the posterior auricularmuscles of SCID mice, as the ear muscle is thin andparticularly advantageous for visualization of the vascu-lar architecture. Vessel morphology was evaluated both2 and 4 wk after implantation, with equivalent results.

Control cells or PDGF-BB alone had no effect onangiogenesis (Fig. 1B, C), while VEGF alone inducedabundant aberrant structures (Fig. 1D), which laterevolved into large angiomas.

We tested whether coimplantation of a mixture ofVEGF- and PDGF-BB-expressing cell populations couldprevent aberrant vessel development and lead to nor-mal angiogenesis. Although highly branched networksof short regular capillaries were induced (Fig. 1E),aberrant structures similar to those observed with VEGFalone could always be found (Fig. 1F, G). To determinewhether the induction of aberrant angiogenesis de-pended on the relative dose of VEGF and PDGF-BB, wevaried the ratio of the two populations in the implantedmixture. V cells were kept constant at 50%, and P cellswere decreased to 20, 10, 5, and 1% (P20, P10, P5 and P1,respectively), with the remainder being made up ofcontrol cells. Alternatively, P cells were maintained at50%, and V cells were similarly decreased to 20, 10, 5,and 1% (V20, V10, V5, and V1, respectively). WhenPDGF-BB-expressing cells comprised just 1% (P1), onlyVEGF-induced aberrant structures were detected. How-ever, as the proportion of P cells increased, the propor-

Figure 1. Uncoordinated expression of VEGF and PDGF-BB leads to a mixture of normal and aberrant vasculature. A) Retroviralconstructs used to generate myoblast populations expressing mVEGF164 (pMFG-V) or hPDGF-BB (pAMFG-P). B–K) At 2 wk afterimplantation in ear muscles (n�7), control cells expressing only CD8 (B) or PDGF-BB (C) induced no angiogenesis. VEGFmyoblasts (D) induced aberrant vessels. A 50:50 mixture of the two populations (E–G) induced robust normal angiogenesis (E)but also aberrant vascular structures (F, arrows; G). Vessels are stained brown after intravascular lectin perfusion and implantedmyoblasts expressing �-galactosidase are stained blue (X-gal). Different proportions of coimplanted cells (n�4), with a 50:1excess of PDGF-BB expression (V1) or 50:20 of VEGF expression (P20), did not prevent the development of aberrantangiogenesis (I, K) in neighboring large areas of normal vessels (H, J). Scale bars � 25 �m.

2489BALANCED VEGF AND PDGF-BB FOR NORMAL ANGIOGENESIS

tion of normal capillary networks increased (Fig. 1H, J),but aberrant structures were still always present, evenwhen only 1% of cells expressed VEGF (Fig. 1I, K).Therefore, irrespective of the ratio of cells expressingVEGF to PDGF-BB, aberrant angiogenesis was consis-tently observed. These findings suggest that normalangiogenesis cannot be reliably achieved when thetwo factors are randomly localized in the cellularmicroenvironment due to secretion by distinct mus-cle cells.

In a second approach designed to ensure colocaliza-tion of VEGF and PDGF-BB, we generated cell popula-tions in which each nucleus expressed both factors.Sequential transduction with two independent con-structs ensured coexpression of the factors in each cell,but at random ratios due to differential vector uptake(Fig. 2A). The resulting population produced similaramounts of VEGF as V cells (132.71.7 ng/106 cells/d)and of PDGF-BB as P cells (54.63.4 ng/106 cells/d).After in vivo implantation, coexpression of the twofactors at random ratios yielded extensive orderly cap-

illary networks, but aberrant angioma-like structureswere also always present (Fig. 2B, C).

Coordinated expression of VEGF and PDGF-BB usinga bicistronic vector produces uniformly normal andmature vessels

In a third approach, we aimed to achieve colocalizedexpression of both factors in fixed relative amounts(coordinated expression). Myoblasts were transducedwith a bicistronic construct [designated VIP for VEGF–internal ribosomal entry site (IRES)–PDGF-BB; Fig.2D], which ensures the translation of both sequencesfrom a single mRNA (22). Two cell populations weregenerated by different numbers of transduction rounds(VIPlow and VIPhigh). VIPlow cells expressed 58.9 5.8ng of VEGF and 9.7 0.7 ng of PDGF-BB/106 cells/d.VIPhigh cells expressed 107.9 6.2 ng of VEGF and26.5 1.6 ng of PDGF-BB/106 cells/d. FACS analysisshowed that transgene expression in all populationswas heterogeneous on a cell-by-cell basis, as evidenced

Figure 2. Coordinated expression of VEGF and PDGF-BB induces only homogeneous normal vessels. A) Myoblasts coexpressingVEGF and PDGF-BB in each cell, but at random relative levels, were generated by sequential transduction with two separateretroviral constructs. B, C) At 2 wk after implantation (n�5), coexpression at random relative levels induced some normalcapillaries (B) but also aberrant bulbous vascular structures (C). D) The pAMFG-VIP bicistronic construct ensured coordinatedexpression of VEGF and PDGF-BB at a fixed ratio in the microenvironment around each transduced cell. E–G) Coordinatedcoexpression induced only robust normal angiogenesis (n�10). Arrows (G) indicate sprouting capillaries. H, I) Amount ofangiogenesis, assessed as VLD (H), and distribution of vessel diameters (I) were quantified in areas implanted with control CD8,P, V, or VIPhigh myoblasts (n�4–5). VLD � vessel length (mm)/area of effect (mm2). Scale bars � 25 �m. ***P 0.001.

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by staining for the coexpressed cell-surface markerCD8a (data not shown), which has been shown tocorrelate directly with the amount of transgene produc-tion in individual cells (23). Following implantationinto muscles, coordinated coexpression by both VIPpopulations induced a well-structured vasculature, withhighly branched networks of homogeneously sized cap-illaries in all sites (Fig. 2E–G). Remarkably, even thoughVIPhigh cells expressed heterogeneous VEGF levels sim-ilar to the V population, no aberrant structures wereever observed.

As a measure of the amount of vasculature induced,VLD was determined in the implanted muscles. Theresults for this and subsequent analyses are shown forVIPhigh cells, although VIPlow implantations yieldedequivalent findings. PDGF-BB alone did not increasethe amount of vasculature. VEGF alone led to some-what reduced VLD due to the replacement of normalcapillaries with hyperfused bulbous structures (ref. 24and Fig. 2H). By contrast, coordinated coexpression ledto a 75% increase in VLD compared with controls.Thus, the extent of vasculature induced was greatlyincreased by VIP cells.

Vessel diameter distribution is a quantitative mea-sure of vessel size homogeneity, a feature of normalvasculature. As shown in Fig. 2I, normal capillaries incontrol areas were homogeneous, with a mediandiameter of 6.8 �m. PDGF-BB alone caused a non-significant enlargement (median diameter 7.7 �m,P�n.s.) with no increase in the range of size distri-bution relative to controls. By contrast, VEGF-in-duced vessels not only were substantially dilated(median diameter 21.8 �m, P0.001) but also exhib-ited a marked increase in size heterogeneity. Notably,coexpression of VEGF and PDGF-BB by VIPhigh cellsprevented aberrant dilation and led to only amoderate increase in the median vessel diameter to8.7 �m (P0.05), while preserving size homo-geneity.

To determine whether the vessels induced by VIPcells were mature, we evaluated whether they acquired

pericytes necessary for stabilization (25). Normal mus-cle capillaries were uniformly covered with pericytes,which expressed NG2 and lacked �SMA (Fig. 3A). Bycontrast, the aberrant structures induced by VEGFalone lacked typical pericytes and were coated by athick smooth muscle layer (Fig. 3B). PDGF-BB aloneled to the accumulation of NG2-positive pericytes be-tween muscle fibers, in accordance with previous re-ports (26), but no increase in vasculature (Fig. 3C). Bycontrast, coordinated expression of both factors by VIPcells led to coverage of new vessels with normal peri-cytes, which were also found to be embedded in thevascular basement membrane, in close contact with theendothelium (Fig. 3D, E).

PDGF-BB coexpression via a bicistronic vector doesnot affect transient induction of vascular leakage byVEGF

An increase in endothelial permeability is an intrinsicproperty of VEGF-induced angiogenesis. PDGF-BB ex-pression did not increase plasma extravasation abovebasal levels at any time point (Fig. 4A), whereas, as wepreviously observed (3), VEGF-induced vascular leak-age was transient, peaked at 4 d, and subsided by 2 wk.PDGF-BB coexpression did not inhibit the initial surgein VEGF-induced vascular leakage, which also de-creased at 1 and 2 wk. As total plasma leakage dependsdirectly on the surface of induced vasculature, the totalamount of extravasated Evans blue (ng) was normal-ized to the total vessel surface (mm2) in the areas ofeffect 2 wk after implantation of CD8, P, V, or VIPhigh

myoblasts. As shown in Fig. 4B, extravasation fromvessels induced by both VEGF alone and together withPDGF-BB was actually less than from normal vessels inareas implanted with control and PDGF-BB-expressingmyoblasts (Fig. 4B). These results show that VEGF-induced leakage is transient and is not affected byPDGF-BB coexpression.

Figure 3. PDGF-BB restores normal pericytecoverage to VEGF-induced vessels. Vessels in-duced by implantation of control CD8 (A), V(B), P (C), or VIPhigh myoblasts (D, E; n�3) inhindlimb tibialis anterior muscles were immu-nostained with antibodies against CD31 (endo-thelium, green), NG2 (pericytes, red), and�SMA (smooth muscle cells, blue in A–D), orlaminin (basal lamina, blue in E) on frozensections. Arrows (A, D, E) indicate pericyteswith typical branched processes. Asterisks (B)indicate the lumen of an angioma-like structuredevoid of pericytes and covered with smoothmuscle cells. Boxed area in D is enlarged in E. Pcells caused proliferation of pericytes betweenmuscle fibers, but no angiogenesis (C), whileVIP myoblasts induced a network of pericyte-covered branched capillaries (D, E). Scale bars �25 �m.

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PDGF-BB delivered via a bicistronic vectornormalizes the remodeling of VEGF-induced vessels

To understand how PDGF-BB prevented the genera-tion of angioma-like structures by uncontrolled VEGFexpression, we investigated the early stages of vasculargrowth after factor delivery. A time course (Fig. 5)revealed that, 4 d after myoblast implantation, theinitial response to VEGF overexpression was a markedcircumferential enlargement of preexisting vessels, sim-ilar to the previously described “mother” vessels (ref. 27and Fig. 5C), that were almost devoid of pericytes (Fig.5E) and rapidly remodeled into bulbous smooth mus-cle-covered structures by 7 d (Fig. 5G). PDGF-BB coex-

pression did not affect initial vessel enlargement byVEGF (Fig. 5D), but it prevented the early loss ofpericytes, which instead accumulated in the interstitialspace between activated vessels by 4 d (Fig. 5F), andconverted vascular remodeling to a network of maturehomogeneous capillaries by 7 d, with pericytes in closecontact with the endothelium (Fig. 5H). Parallel tissuesamples were analyzed for VEGF concentration pertotal amount of protein by ELISA at the same timepoints. Notably, muscle tissues transplanted with VIPmyoblasts expressed up to 2-fold more VEGF in vivothan V cells at 4 and 7 d post-transplant (253.024.8 vs.193.815.5 pg/mg protein at 4 d and 264.48.0 vs.110.516.2 pg/mg protein at 7 d, respectively;P0.001). These data show that PDGF-BB expressed ina fixed ratio using a bicistronic vector normalizes theeffects of VEGF even when expressed at high levels.

The balance between VEGF and PDGF-BB dosageregulates the switch between normal and aberrantangiogenesis

To further assess whether the induction of normal oraberrant angiogenesis by specific VEGF doses dependson PDGF-BB signaling, we performed loss- and gain-of-function experiments (Fig. 6). Specific low and highVEGF levels (70 and 120 ng/106 cells/d, respectively)were homogeneously expressed by clonal populationsof transduced myoblasts, in which every cell producedthe same amount of factor. These populations inducednormal and aberrant angiogenesis respectively (Fig. 6B,E), as described previously (3). In a loss-of-functionapproach, blockade of endogenous PDGF-BB wasachieved by coexpression of a truncated soluble form ofPDGF receptor-� (sPDGFR-�; ref. 28). In the presenceof low VEGF levels, expression of sPDGFR-� led to

Figure 4. VEGF-induced vascular leakage is transient and notaffected by PDGF-BB. A) Vascular leakage was measured 4, 7,and 14 d after implantation of control cells (CD8), P, V, orVIPhigh (VIP) myoblasts in ear muscles and is expressed asnanograms of extravasated Evans blue per milligram of tissue(n�5). Both V and VIP cells caused a transient increase inleakage that peaked at 4 d and returned to baseline by 14 d.B) Total leakage was normalized to the amount of inducedvasculature 2 wk after implantation of the same populations(nanograms of extravasated Evans blue per square millimeterof total vessel surface; n�5), showing that both V- andVIP-induced vasculature was less permeable than controlvessels. *P 0.05; **P 0.01.

Figure 5. PDGF-BB regulates vascular remodeling by early pericyte recruitment. A–D) Blood vessel morphology was analyzed bywhole-mount lectin staining 4 d after implantation with control CD8 (A), P (B), V (C), or VIPhigh myoblasts (D). E–H) Tibialisanterior muscle sections were immunostained 4 d (E, F), and 7 d (G, H) after implantation with V (E, G), or VIPhigh myoblasts(F, H) with antibodies against CD31 (endothelium, red), NG2 (pericytes, green), and �SMA (smooth muscle cells, blue).PDGF-BB coexpression did not affect initial vascular enlargement by VEGF but induced early pericyte recruitment by 4 d, andby 7 d caused remodeling to normal pericyte-covered vessels instead of aberrant structures. Asterisk (G) indicates the lumen ofan aberrant angioma-like structure (n�4 in all cases).

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many aberrant structures in all implantation sites (Fig.6A), similar to those induced by high VEGF alone (Fig.6E). In addition, angioma-like angiogenesis induced byhigh VEGF levels developed faster and more extensivelywhen sPDGFR-� was expressed (Fig. 6D). In gain-of-function experiments, we used clonal populations de-rived from the heterogeneous VIP population de-scribed in Fig. 2, which expressed similar low and highVEGF levels to those of the V clones (VIPlow: 74 ngVEGF/106 cells/d; VIPhigh: 141 ng VEGF/106 cells/d).Coexpression of exogenous PDGF-BB did not affectnormal angiogenesis induced by low VEGF alone (Fig.6C), but completely prevented the aberrant structuresinduced by high VEGF alone, yielding uniformbranched capillary networks (Fig. 6F), confirming theresults seen with the heterogeneous populations (Fig.2). Expression of sPDGFR-� (Fig. 6G), like control cells(Fig. 6H) or PDGF-BB alone (Fig. 6I), did not affectpreexisting vascular networks. Together, these resultsshow that the induction of normal or aberrant angio-genesis depends on the relative dosage of VEGF andPDGF-BB and consequent signaling, rather than on aspecific VEGF dose.

Coordinated expression of VEGF and PDGF-BB bybicistronic delivery leads to functional collaterals andreversal of ischemia

To test the efficacy of balanced coexpression of VEGFand PDGF-BB, we tested the functional effects of VIPcell implantation in a hindlimb ischemia model. Thedistal thigh muscles of SCID mice were injected witheither vehicle or 8 � 106 myoblasts expressing �-galac-tosidase, VEGF alone, PDGF-BB alone, or the VIPbicistronic vector encoding both factors immediatelyafter induction of ischemia (22) and analyzed 2 wk

later. H&E staining of ischemic muscles confirmed thepresence of tissue damage, with centralized nuclei inthe myofibers and infiltration of inflammatory cells(data not shown). PDGF-BB cells recruited pericytes,but did not induce angiogenesis (Fig. 7A, B), similar totheir effects when transplanted into nonischemic mus-cles. VEGF cells moderately increased vascular densitycompared to control cells (Fig. 7B), but many vesselswere angioma-like, devoid of pericytes, and encom-passed by smooth muscle cells (Fig. 7A). By contrast,both VIPlow and VIPhigh cell populations led to signifi-cant growth of uniformly normal, pericyte-covered cap-illaries (Fig. 7A, B).

Notably, only VIP cells led to significant functionalimprovement in ischemic muscles. Both efficientangiogenesis and arteriogenesis were detected. Tis-sue perfusion was restored and damage repaired.Blood flow in ischemic muscles (Fig. 7C) was unaf-fected by either control or PDGF-BB-expressing cellsand only moderately increased by VEGF alone. Bycontrast, balanced factor coexpression by VIP cellssignificantly improved perfusion to greater than non-ischemic levels. Similarly, collateral growth was sub-stantially increased only when coexpression oc-curred, and the number of collateral arteriesdoubled (Fig. 7D). Consistent with these angiogeniceffects, while neither VEGF nor PDGF-BB alonegreatly altered the percentage of damaged myofibers,coordinated coexpression by VIP cells led to a re-markable reduction in muscle damage by �50% (Fig.7E). These findings provide compelling evidencethat coordinated coexpression of the two factorsusing the bicistronic VIP vector had a major func-tional impact on ischemic limb muscles, restoringblood flow and reducing tissue damage.

Figure 6. The balance between VEGF andPDGF-BB signaling controls whether angiogen-esis is normal or aberrant. Clonal myoblastpopulations, homogeneously expressing spe-cific low (B) or high (E) VEGF levels, inducednormal and aberrant angiogenesis, respectively.Loss-of-function experiments, in which endog-enous PDGF-BB was blocked by delivery ofsoluble PDGFR-�, led to aberrant angiogenesiseven in the presence of low VEGF levels (A) andaccelerated it in the presence of high VEGFlevels (D). Conversely, gain-of-function experi-ments in which PDGF-BB was coexpressed atcoordinated levels by VIP clones did not affectnormal angiogenesis by low VEGF (C) but ab-rogated the development of aberrant vessels byhigh VEGF (F). Expression of soluble PDGFR-�alone (G) or PDGF-BB alone (I) did not causeany angiogenic effect compared to control cells(H). Arrows (A, E) indicate aberrant angioma-like structures. Scale bars � 25 �m; n �5/group.

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Gene transfer via adenoviral delivery of a bicistronicvector encoding VEGF and PDGF-BB inducesuniformly normal angiogenesis

We sought to broaden our extensive findings using amodel myoblast-mediated gene delivery system to agene delivery system appropriate for clinical translationas a gene therapy approach. Accordingly, we testedwhether coordinated expression of VEGF and PDGF-BBfrom a single bicistronic cassette could lead to normalangiogenesis irrespective of VEGF levels. For this purpose,four different adenoviral vectors were produced, express-ing the same cassettes as the retroviral vectors used togenerate the CD8, V, P, and VIP myoblast populationsdescribed above, and were named Ad-CD8, Ad-VEGF,Ad-PDGF, and Ad-VIP, respectively. Effective growth fac-tor production by the recombinant adenoviruses was

confirmed by ELISA on supernatants after in vitro infec-tion of HEK293 cells.

We injected 1 � 108 infectious units of each virus intothe posterior auricular muscles of SCID mice. Intravas-cular lectin staining 2 wk later showed that bothAd-CD8 and Ad-PDGF vectors did not affect the preex-isting vascular networks (Fig. 8A, B), whereas Ad-VEGFcaused the widespread appearance of aberrant angioma-like structures of heterogeneous sizes and irregularshapes (Fig. 8C), similar to those induced by myoblastsexpressing VEGF alone. By contrast, coordinatedPDGF-BB expression by the Ad-VIP bicistronic vectorprevented all instances of aberrant angiogenesis andinduced only the growth of homogeneous normalcapillaries (Fig. 8D), in agreement with the myoblast-mediated gene delivery system.

These experiments were corroborated in the limb

Figure 7. Coordinated expression of VEGF and PDGF-BB reverses hindlimb ischemia. Ischemic hindlimbs of SCID mice weretreated with vehicle (BSA) or myoblasts expressing only �-galactosidase (LacZ), PDGF-BB, VEGF, or both at coordinated levels(VIPlow and VIPhigh, expressing two different average VEGF levels). Angiogenic response (A, B) and regional blood flow (C)were assessed on the distal thigh muscles (quadriceps femoris and biceps femoris), where cells were injected. Collateral growth(D) was assessed in the proximal adductor muscle, which is anatomically remote from the injection sites, and muscle damage(E) was determined in the calf muscles (tibialis anterior and gastrocnemius), where ischemia was maximal. A) Immunostainingof vessels in implanted muscles with antibodies against endothelial CD31 (PECAM; red), pericyte NG2 (green), or smoothmuscle �SMA (blue). Scale bar � 50 �m; n � 5–7. B) VLD quantification (micrometers of vessel length per muscle fiber in thearea of effect, n�5–7) in the treated muscles (blue bars) and contralateral nonischemic muscles (white bars). C) Relative bloodflow in ischemic muscles (percentage of the contralateral nonischemic leg, n�5–7). Dotted line represents normal flow (100%).D) Number of collaterals in the proximal adductor muscle group of treated legs (n�3). E) Damaged tissue in ischemic calfmuscles of treated limbs (percentage area of the total muscle on histological sections, n�5–6). *P 0.05; **P 0.01.

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muscles by injection of the viral vectors into the tibialisanterior. Immunostaining of frozen tissue sectionsshowed that the Ad-PDGF vector did not induce anyangiogenesis above that of the control Ad-CD8 vector(Fig. 8E, F). Aberrant vasculature was induced by theAd-VEGF vector (Fig. 8G). By contrast, Ad-VIP vectorinjection caused the efficient growth of mature, peri-cyte-covered capillaries (Fig. 8H) and prevented theappearance of aberrant, smooth muscle-covered angio-ma-like structures. These data lend further support tothe finding that gene therapy via adenoviral delivery ofa bicistronic vector encoding both VEGF and PDGF-BBto muscles of the leg, which are the targets of ischemia,leads to normal angiogenesis.

DISCUSSION

An unresolved therapeutic challenge is to achieve ef-fective induction of angiogenesis leading to reversal oflimb ischemia by growth factor delivery, a problem ofmajor magnitude in the United States, as �100,000limb amputations occur every year due to untreatableperipheral vascular disease (1). Using our myoblast-mediated gene delivery model system, which facilitatestesting of variables in a well-controlled manner, wepreviously showed that low-level VEGF expression givesrise to orderly vasculature, whereas high-level VEGFleads to aberrant structures that eventually evolve intohemangiomas (3, 5). Here we demonstrate that theinduction of normal or aberrant angiogenesis dependson the relative expression levels of VEGF and PDGF-BB,rather than on a specific amount of VEGF. Balancedcoexpression of VEGF and PDGF-BB in a fixed relative

ratio was achieved using a single bicistronic vector andresulted in homogeneously normal angiogenesis, irre-spective of VEGF levels. Notably, the results obtainedusing two independent bicistronic gene delivery plat-forms were similar. The beneficial effects on angiogen-esis of retrovirally transduced myoblasts were corrobo-rated by a clinically relevant gene therapy approachentailing direct injection into muscles of adenoviralvectors.

Taking advantage of the high degree of controlafforded by myoblast-based gene transfer, we testedvarious means of delivering the two factors: high-levelexpression of VEGF and PDGF-BB in separate popula-tions of juxtaposed cells; different ratios of these cellpopulations skewed either toward VEGF or PDGF-BB;or high-level coexpression of VEGF and PDGF-BB ineach cell by sequential transduction of two differentvectors. In each case, although a remarkable network oforderly vasculature resulted, which was far better thanthat seen with VEGF alone, aberrant angioma-likevessels were also detected. In agreement with ourfindings with myoblast-mediated gene delivery, codeliv-ery of VEGF and PDGF-BB by two separate adenoviralvectors led to angiogenesis with defective pericyte re-cruitment (26). We hypothesized that absolute levels ofthe two factors were not as important as the relativedosage present in the microenvironment of the grow-ing vessels, so that both endothelial cells and pericyteswould be exposed to similar balanced growth factorstimulation. A single bicistronic vector, VIP, ensuredboth colocalization and a fixed ratio of VEGF toPDGF-BB around each cell (coordinated coexpression)when delivered either by myoblasts or by adenoviralvectors. The data presented here show that, irrespective

Figure 8. Adenoviral delivery of a single bicistronic vector leads to coordinated expression of VEGF and PDGF-BB and preventsaberrant angiogenesis. Adenoviruses expressing PDGF-BB (Ad-PDGF), VEGF (Ad-VEGF), or both (Ad-VIP) were injected in ear(A–D) or tibialis anterior muscles (E–H) of immunocompromised mice. Adenovirus expressing only CD8 (Ad-CD8) was used asa control. After 2 wk, blood vessels were visualized in ear whole-mount preparations after intravascular lectin perfusion (inbrown; A–D), or after immunofluorescent staining of endothelium (CD31, in red), pericytes (NG2, in green), and smoothmuscle cells (�-SMA, in white) in cryosections of hindlimb muscles (E–H). In both locations, aberrant angioma-like structuresinduced by Ad-VEGF (C, G) were prevented by coexpression of VEGF and PDGF-BB by Ad-VIP, which induced only normalpericyte-covered capillaries (D, H). Scale bars � 20 �m.

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of the markedly different absolute levels of the twofactors expressed, two independent VIP cell popula-tions induced a large increase in histologically normaland mature vasculature. Notably, this balanced, spa-tially and temporally coordinated expression of bothgrowth factors led to an increase in functional bloodvessels, efficient collateral arteriogenesis, reduced mus-cle damage, and correction of muscle ischemia. Toensure that effective muscle perfusion was measured,rather than effects on the superficial skin layer, bloodflow was determined by fluorescent microsphere count-ing in histological sections, as this method representsthe gold standard and is routinely used to validateresults obtained with new techniques (29).

VEGF has been shown to negatively regulate pericytefunction by inhibiting PDGFR� phosphorylationthrough the formation of a nonfunctional VEGFR2/PDGFR� complex (19). Consistent with this report, wefound that VEGF expression alone leads to initial vesselenlargement with an almost complete loss of pericytes.Our data extend these findings by demonstrating thatthe antipericyte effect of VEGF can be completelyovercome by balanced coexpression of PDGF-BB in thesame cells, preventing pericyte loss at 4 d and produc-ing homogeneous capillary networks at 7 d. Thus, ourdata highlight that balanced levels and spatial colocal-ization of VEGF and PDGF-BB are both essential foroptimal therapeutic benefit. Furthermore, although inphysiological angiogenesis PDGF-BB is produced byendothelial cells (30–32), our results show that this cellsource is not essential for normal vessel formation,provided that VEGF and PDGF-BB are produced inspatiotemporally colocalized gradients and balancedamounts. In fact, it should be noted that, contrary toprevious observations of inadequate pericyte coverageon codelivery by two separate adenoviral vectors (26),coordinated coexpression from a single bicistronicadenovirus both restored physiological pericyte recruit-ment and ensured complete normalization of newlyinduced vasculature. These findings suggest that thisbalance is necessary and sufficient for effective thera-peutic angiogenesis in ischemic muscle.

In the present experiments, VEGF and PDGF-BBwere expressed simultaneously by the bicistronic vector.Because of the success of this approach, we did notdetermine whether sequential expression of the twogrowth factors was even more efficacious than simulta-neous expression. Nonetheless, our findings suggestthat early expression of PDGF-BB did not limit VEGF-driven capillary growth. The larger amount of vesselgrowth found with synchronous expression of the twogrowth factors, compared to expression of VEGF alone,argues that increased pericyte recruitment within thefirst 4 d did not have a net negative effect on angiogen-esis.

Critical to our findings was the delivery of the angio-genic factors to skeletal muscle, the clinically relevanttarget for treatment of peripheral ischemia. Othershave studied VEGF and PDGF-BB interaction in non-muscle tissues and observed different effects. Although

PDGF-BB is proangiogenic in the chicken chorioallan-toic membrane and in Matrigel plug assays (19), it doesnot induce angiogenesis either in normal or ischemicskeletal muscle (26). Furthermore, PDGF-BB expres-sion in the cornea does not correct the aberrantfeatures of VEGF-induced vessels (18), by contrast withits effects in skeletal muscle. Thus, these growth factorsare context-dependent and their effects cannot bereadily extrapolated across tissues.

Most striking was the functional impact of coordi-nated VEGF and PDGF-BB delivery to ischemic limbmuscles of mice. The extent and morphology of theinduced vasculature caused not only increased angio-genesis but also a doubling of collateral arteries. This,in turn, led to increased tissue perfusion and substan-tially reduced muscle damage assessed by myofiberintegrity. All of these functional changes were observedonly with VIP cells, not V, P, or control cells.

Our results have major implications for therapeuticangiogenesis. VEGF gene delivery has a narrow thera-peutic window for the treatment of muscle ischemia(16, 17), which has hindered use of gene therapyvectors (2). Based on our findings using adenoviralvectors, we predict that balanced expression of VEGFand PDGF-BB in the same cells using viral delivery of asingle bicistronic vector will overcome this problem,enabling safe and efficacious gene therapy for angio-genesis in patients with limb ischemia.

The authors thank N. Di Maggio (Basel University Hospi-tal, Basel, Switzerland) for critical comments on the manu-script, P. Lindblom (Karolinska Institute, Stockholm, Swe-den) for the hPDGFb cDNA, and L. T. Williams (University ofCalifornia, San Francisco, CA, USA) for the sPDGFR-� cDNA.This work was supported by an American Heart AssociationScientist Development grant (043031), a Swiss National Sci-ence Foundation grant (310030-127426), and the EU FP7grant ANGIOSCAFF (CP-IP 214402) to A.B.; by a Deutsche-Forschungsgemeinschaft grant (DE 740/1-1) to G.V.D.; byU.S. National Institutes of Health (NIH) grants HL-24136,HL-59157 and CA-082923 to D.M.M.; and by NIH grantsAG-009521, HL-065572, AG-020961, and AG-024987 and sup-port from the Baxter Foundation to H.M.B. The authorsdeclare no conflicts of interest.

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Received for publication November 11, 2011.Accepted for publication February 21, 2012.

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