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Adenovirus-Mediated VEGF121 Gene Transfer Stimulates Angiogenesis in Normoperfused Skeletal Muscle and Preserves Tissue Perfusion After Induction of Ischemia

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Adenovirus-Mediated VEGF121 Gene Transfer Stimulates Angiogenesis in Normoperfused Skeletal Muscle and Preserves Tissue Perfusion After Induction of Ischemia
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  Mark Talan and Maurizio C. CapogrossiMassimiliano Marrocco-Trischitta, Edward G. Lakatta, Piero Anversa, Richard G. S. Spencer,Fishbein, Antonella Zacheo, Roberta Palumbo, Stefania Straino, Costanza Emanueli, Luis Henrique W. Gowdak, Lioubov Poliakova, Xiaotong Wang, Imre Kovesdi, Kenneth W. IschemiaNormoperfused Skeletal Muscle and Preserves Tissue Perfusion After Induction of Gene Transfer Stimulates Angiogenesis in121Adenovirus-Mediated VEGF Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2000 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Circulation doi: 10.1161/01.CIR.102.5.5652000;102:565-571 Circulation. http://circ.ahajournals.org/content/102/5/565 World Wide Web at: The online version of this article, along with updated information and services, is located on the  http://circ.ahajournals.org//subscriptions/ is online at: Circulation Information about subscribing to Subscriptions:  http://www.lww.com/reprints Information about reprints can be found online at: Reprints:  document. Permissions and Rights Question and Answer this process is available in theclick Request Permissions in the middle column of the Web page under Services. Further information aboutOffice. Once the online version of the published article for which permission is being requested is located, can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Circulation in Requests for permissions to reproduce figures, tables, or portions of articles srcinally published Permissions:  by guest on March 12, 2014http://circ.ahajournals.org/ Downloaded from by guest on March 12, 2014http://circ.ahajournals.org/ Downloaded from   Adenovirus-Mediated VEGF 121  Gene Transfer StimulatesAngiogenesis in Normoperfused Skeletal Muscle andPreserves Tissue Perfusion After Induction of Ischemia Luis Henrique W. Gowdak, MD; Lioubov Poliakova, MD; Xiaotong Wang, MD; Imre Kovesdi, PhD;Kenneth W. Fishbein, PhD; Antonella Zacheo, BS; Roberta Palumbo, PhD; Stefania Straino, BS;Costanza Emanueli, PhD; Massimiliano Marrocco-Trischitta, MD; Edward G. Lakatta, MD;Piero Anversa, MD; Richard G.S. Spencer, MD, PhD; Mark Talan, MD, PhD; Maurizio C. Capogrossi, MD  Background  —Administration of angiogenic factors stimulates neovascularization in ischemic tissues. However, there is noevidence that angiogenesis can be induced in normoperfused skeletal muscles. We tested the hypothesis thatadenovirus-mediated intramuscular (IM) gene transfer of the 121-amino-acid form of vascular endothelial growth factor(AdCMV.VEGF 121 ) could stimulate neovascularization in nonischemic skeletal muscle and consequently attenuate thehemodynamic deficit secondary to surgically induced ischemia.  Methods and Results —Rabbits and rats received IM injections of AdCMV.VEGF 121 , AdCMV.Null, or saline in the thigh,4 weeks (rabbits) or 2 weeks (rats) before femoral artery removal in the injected limb. In unoperated rats, at the site of injection of AdCMV.VEGF 121 , we found 96% and 29% increases in length density of arterioles and capillaries,respectively. Increased tissue perfusion (TP) to the ischemic limb in the AdCMV.VEGF 121  group was documented, asearly as day 1 after surgery, by improved blood flow to the ischemic gastrocnemius muscle measured by radioactivemicrospheres (AdCMV.VEGF 121  5.69  0.40, AdCMV.Null  2.97  0.50, and saline  2.78  0.43 mL    min  1   100 g  1 , P  0.001), more angiographically recognizable collateral vessels (angioscore) (AdCMV.VEGF 121  50.58  1.48,AdCMV.Null  29.08  4.22, saline  11.83  1.90,  P  0.0001), and improvement of the bioenergetic reserve of thegastrocnemius muscle as assessed by  31 P NMR spectroscopy. Follow-up studies showed that superior TP to the ischemiclimb in the AdCMV.VEGF 121  group persisted until it was equalized by spontaneous collateral vessel development inuntreated animals. Conclusions —IM administration of AdCMV.VEGF 121  stimulates angiogenesis in normoperfused skeletal muscles, and thenewly formed vessels preserve TP after induction of ischemia.  ( Circulation . 2000;102:565-571.)Key Words:  angiogenesis    endothelium-derived factors    genes    ischemia    peripheral vascular disease T reatment of patients suffering from peripheral arterial oc-clusive disease remains a considerable clinical challengedespite advances in both surgical and percutaneous revascular-ization techniques. Many patients cannot benefit from thesetherapies because of the anatomic extent and the distribution of arterial occlusion. In such patients, new therapeutic strategieshave been sought to prevent the development of disablingsymptoms related to ischemia, eg, claudication, resting pain, andloss of tissue integrity in the distal limbs.Recently, therapeutic angiogenesis 1 has been successfullyapplied in animal models of limb 2,3 or myocardial 4,5 ischemia.Phase 1 clinical trials of angiogenesis are under way fortreatment of patients with peripheral vascular obstruction 6 orcoronary artery disease. 7–9 The presence of persistent tissue ischemia at the time of administration of an angiogenic factor has been considered anessential precondition for angiogenic effects. 10 Whether anangiogenic factor delivered to a normoperfused skeletalmuscle before the occurrence of ischemia could stimulateneovascularization and preserve blood perfusion once ische-mia develops remains an unsolved issue. In addition to itsimportance for understanding the basic mechanisms of ther- Received December 21, 1999; revision received March 1, 2000; accepted March 2, 2000.From the Gene Therapy Unit, Laboratory of Cardiovascular Science (L.H.W.G., L.P., X.W., E.G.L., M.T.) and Nuclear Magnetic Resonance Unit,Laboratory of Cellular and Molecular Biology (K.W.F., R.G.S.S.), Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Md; GenVec Inc, Rockville, Md (I.K.); Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata, Istituto di Ricoveroe Cura a Carattere Scientifico, Rome, Italy (A.Z., R.P., S.S., C.E., M.M.-T., M.C.C.); and Department of Medicine, New York Medical College, Valhalla(P.A.). Drs Spencer, Talan, and Capogrossi contributed equally to this article.Presented in part at the 48th Annual Scientific Session of the American College of Cardiology, New Orleans, La, March 8, 1999, and publishedpreviously in abstract form (  J Am Coll Cardiol.  1999;33(suppl A):250A.Correspondence to Mark Talan, MD, PhD, Laboratory of Cardiovascular Science, Gerontology Research Center, 5600 Nathan Shock Dr, Baltimore,MD 21224. E-mail talanm@grc.nia.nih.gov© 2000 American Heart Association, Inc. Circulation  is available at http://www.circulationaha.org  565 Basic Science Reports  by guest on March 12, 2014http://circ.ahajournals.org/ Downloaded from   apeutic angiogenesis, this question is of substantial clinicalsignificance because the majority of patients with peripheralarterial disease do not have chronic ischemia but ratherrecurrent episodes of ischemia during physical activity. 11 The present study was designed to determine whetherprophylactic, adenovirus-mediated gene transfer of the 121-amino-acid form of vascular endothelial growth factor(AdCMV.VEGF 121 ) could stimulate angiogenesis in a nor-moperfused limb and preserve blood flow to the skeletalmuscles after the induction of ischemia, thereby amelioratingthe hemodynamic deficit in the acutely ischemic limb. Methods Subjects The care of animals in this study complied with the  Guide for theCare and Use of Laboratory Animals  (Institute of Laboratory AnimalResources, Commission on Life Sciences, National Research Coun-cil, Washington, DC: National Academy Press, 1996), and allprotocols were approved by the Gerontology Research Center and bythe Istituto Dermopatico dell’Immacolata Animal Care and UseCommittee. Six-month-old male New Zealand White rabbits, weight3.5 to 4.5 kg, and 10-month-old male Wistar rats, weight 500 to600 g, were used. Experimental Design  Adenovirus Vectors Replication-deficient recombinant Ad vectors containing cDNAfor VEGF 121  were engineered according to a technique previouslydescribed 12 and were supplied by GenVec, Inc. Briefly, theAdCMV.VEGF 121  is an E1a  , partial E1b  , partial E3  Ad vectorthat carries, in the E1 position, an expression cassette containing thecytomegalovirus (CMV) immediate early promoter/enhancer drivingthe cDNA for the 121-residue form of human VEGF. AdCMV.Null,used as a control vector, is similar to AdCMV.VEGF 121  but has nogene in the expression cassette.  Intramuscular Administration of AdCMV.VEGF 121 Four weeks before the induction of ischemia, rabbits wererandomly assigned to receive AdCMV.VEGF 121  (10 6 or 10 8 pfu/mL), AdCMV.Null (10 8 pfu/mL), or saline. Rats receivedinjections of AdCMV.VEGF 121  (2  10 9 pfu/mL) or AdCMV.Null(2  10 9 pfu/mL) 2 weeks before surgery. The Ad vectors werestored in dialysis buffer solutions at   70°C. Each solution forinjection was prepared immediately before use and given intra-muscularly in 4 different sites in the thigh (250   L/injection, 1mL total volume in rabbits; 125   L/injection, 0.5 mL totalvolume in rats) along the projection of the femoral artery.  Animal Model of Hindlimb Ischemia Both species underwent surgical procedures to induce unilateral hind-limb ischemia as previously described. 13 In rats, surgery was performedunder intraperitoneal anesthesia with ketamine (60 mg/kg) and xylazine(10mg/kg).Rabbitswerepreanesthetizedwithketamine(50mg/kg)andxylazine (5 mg/kg), intubated, and mechanically ventilated with amixture of 1.5% isoflurane and oxygen. The femoral artery was thenexcised from its proximal srcin as a branch of the external iliac arteryto the point distally where it bifurcates into the saphenous and poplitealarteries. Study Parameters in Rabbits Calf Blood Pressure Ratio For 12 weeks after surgery, calf blood pressure was measuredweekly with a Doppler flowmeter (Vascular Mini-Laboratory III,Parks Medical Electronics) in both hindlimbs of 32 rabbits. 14 Allmeasurements were performed by a single observer blinded to thetreatment regimen. The calf blood pressure ratio (BPR) was definedfor each rabbit as the ratio of systolic pressure of the ischemic limbto systolic pressure of the normal limb.  Blood Flow Measurements Regional blood flow (RBF) to skeletal muscles in both hindlimbs of 64 rabbits was measured in the resting state by use of radioactivemicrospheres at day 1 and then at weeks 1, 4, and 12 after surgery.After animals were placed under anesthesia with pentobarbital (10 to20 mg/kg IV), the chest was opened, and 3.3  10 6 radioactivemicrospheres (15.5   m in diameter) labeled with  51 Cr (NEN LifeScience Products) were injected into the left ventricle. A bloodreference sample was withdrawn from a catheter (18 gauge,Abbocath-T) advanced through the left carotid artery into thedescending aorta at a constant rate of 2 mL/min starting 30 secondsbefore and continuing for 90 seconds after the injection wascompleted. Animals were killed, and the adductor and gastrocnemiusmuscles of both limbs were removed. Tissue samples and referenceblood samples were digested with potassium hydroxide and thenfiltered with glass microfiber filters with 1.6-  m-diameter pores(Whatman International Ltd). The radioactivity of each sample of filter-entrapped microspheres was determined with a liquid scintil-lation counter (model LS5801, Beckman Coulter, Inc). The RBF(mL    min  1   100 g  1 ) was calculated by the formula    T  100  (withdrawal rate/tissue weight)  (cpm tissue  /cpm reference blood ), where cpmis counts per minute.  Postmortem Contrast Angiograms Postmortem contrast angiograms of the ischemic limbs were ob-tained at day 1 after surgery in 15 rabbits. A total of 5 mL contrastmedium (Hypaque sodium 50%, diatrizoate sodium, Nycomed, Inc)was injected into the right common iliac artery at a constant rate of 20 mL/min. Serial images of the ischemic hindlimb were recorded(Digimax MP4000 Series III Workstation, Acoma Medical Imaging,Inc). Collateral vessel development in the thigh was assessed by useof a grid overlay with 2-mm squares. The angiographic score(angioscore) was defined as the total number of contrast-opacifiedvessels crossing the squares divided by the total number of squaresin the ischemic thigh multiplied by 100. All countings were per-formed by a single observer blinded to the treatment regimen. Wealso performed a qualitative assessment by observation of the arterialfilling in the distal leg (saphenous and popliteal arteries). Forpurposes of comparison among different treatment groups, thearterial filling was noted as present or absent. Study Parameters in Rats Western Blot Analysis and ELISA VEGF expression in rat skeletal muscles was examined by Westernblot analysis and ELISA (R&D Systems, Inc). 10  31  P NMR Spectroscopy 31 P NMR spectroscopy was used to determine bioenergetic charac-teristics of the gastrocnemius muscles at rest and during electricalstimulation. Studies were conducted in 11 rats on days 1, 7, and 14after surgery. After sedation with ketamine (60 mg/kg) and xylazine(10 mg/kg), platinum stimulation electrodes (Grass InstrumentsManufacturing) were inserted into the proximal head of the gastroc-nemius and in the Achilles’ tendon and connected to a programmablestimulator (model S-10, Grass Instruments Manufacturing) with anisolation transformer via a low-pass filter. An elliptical radiofre-quency surface coil tuned to the  31 P resonance was positioned on thegastrocnemius. 15 The foot of the stimulated leg was tied to astrain-gauge force transducer (Grass Instruments Manufacturing)with a 3-0 silk suture. The transducer was connected to a strain-gauge conditioner, preamplifier, and chart recorder (Gould Instru-ment Systems, Inc), allowing continuous monitoring of musclecontraction force. Electrical stimulation was applied as train of pulsepairs, each pulse 200   s long, with a 200-ms interval separating theelements of each pair. This was repeated every 2 seconds. Thestimulation voltage was determined by observation of the voltagebeyond which there was no further increase in contraction force.  566 Circulation  August 1, 2000  by guest on March 12, 2014http://circ.ahajournals.org/ Downloaded from   NMR data were acquired on a 1.9-T/31-cm NMR spectrometer(Biospec, Bruker Medizintechnik GmbH). The proton NMR signalfrom the  31 P-tuned coil was detected and used for shimming. 16 Adiabatic excitation pulses were used to compensate for radiofre-quency inhomogeneity. Pulses were applied every 2 seconds, so thatthe 64 transients collected for each spectrum required 2 minutes. Ineach NMR experiment, 1 spectrum was collected immediately beforestimulation, 3 spectra were collected during stimulation, and 6spectra were collected immediately after cessation of stimulation.This protocol was then repeated for the contralateral leg.After line broadening and Fourier transformation, each spectrumwas manually phased and its baseline corrected with a spline fit. Thedata were processed to yield the quantity PCr/(PCr  P i ), where PCris phosphocreatine and P i  is inorganic phosphate, as a function of time before, during, and after stimulation.  Histological and Morphometric Analysis A histological and morphometric analysis was performed on 14 ratsto evaluate the angiogenic effect of AdCMV.VEGF 121  in the absenceof ischemia. Fifteen days after injection of the viral vector, both legswere perfused via the abdominal aorta with 10% buffered formalin at100 mm Hg for 15 minutes. Subsequently, the adductor and gastroc-nemius muscles were immersion-fixed in formalin for 48 hours.From each sample, sections were cut with the muscle fibers orientedtransversely and were stained with smooth muscle   -actin antibodyto identify arterioles and differentiate them from capillaries andveins. Sections were incubated with mouse monoclonal anti–  -smooth muscle actin (clone 1A4, Sigma Chemical Co) diluted 1:30in PBS and subsequently with anti-mouse rhodamine-labeledantibody.For the morphometric analysis, the total area of the muscle presentin each section was examined at  200 magnification. Arteriole andcapillary length density were measured as previously described. 5,17 The measurements were performed by a single observer blinded tothe treatment regimen. Statistical Analysis Results are expressed as mean  SEM. Statistical comparisons wereperformed by 2-factor ANOVA (treatment groups and time pointsafter surgery) (BMDP Statistical Software). For NMR data analysis,1-way ANOVA (treatment groups) for repeated measurements (re-peated recorded spectra) was performed. All data were analyzed formain effects and interaction; simple effects were calculated whenappropriate. The qualitative angiographic data were evaluated withPearson’s    2 test. A value of   P  0.05 was considered statisticallysignificant for main effects and  P  0.01 for interaction and simpleeffects. Results Expression Kinetics of AdCMV.VEGF 121 In AdCMV.VEGF 121 -infected adductor muscles, transgeneexpression was elevated 3 days after infection and returned tocontrol levels at day 14 (Figure 1). ELISA showed that withAdCMV.VEGF 121 , VEGF levels were 3.7  1.1 ng/mg protein3 days after infection (n  3) and were undetectable at day 14(n  3). In AdCMV.Null-infected muscles, VEGF levels wereundetectable at days 3 (n  3) and 14 (n  3).These results are consistent with those of previous in vivostudies that have shown that Ad-mediated transgene expres-sion is transient and ceases within a few days afterinfection. 5,12,18 Calf BPR Calf BPR was similar in all groups 1 week after surgery andimproved in all groups afterward until week 12 ( P  0.0001)(Figure 2). However, animals treated with AdCMV.VEGF 121  at10 8 pfu/mL showed a faster rate of recovery between weeks 1and 4 than both controls ( P  0.0001) and animals treated withAdCMV.VEGF 121  at 10 6 pfu/mL ( P  0.001). The higher BPR inthe AdCMV.VEGF 121  at 10 8 pfu/mL group remained significantuntil week 8. In animals treated with AdCMV.VEGF 121  at 10 6 pfu/mL, the rate of recovery was not different from that of controls. Figure 1.  Transgene expression in rat adductor muscles. At day3 after infection, Western analysis shows evidence of VEGFexpression in muscles injected with AdCMV.VEGF 121 , whereasat day 14, VEGF levels are back to control and comparable tothose in muscles injected with AdCMV.Null. Each point repre-sents a different animal, and similar results were obtained in 3separate experiments. Figure 2.  BPR as a function of time aftersurgery. Animals treated with a lower con-centration of AdCMV.VEGF 121  (10 6 pfu/mL)had a rate of recovery similar to both con-trol groups. However, BPR in AdCMV-.VEGF 121  at 10 8 pfu/mL group was signifi-cantly higher than AdCMV.VEGF 121  at 10 6 pfu/mL and both control groups until week8. Pretreatment with AdCMV.VEGF 121  at 10 8 pfu/mL clearly accelerated tissue perfusionrecovery, especially between weeks 1 and4 after induction of ischemia. * P  0.0001 vsall other groups. Gowdak et al Prophylactic Angiogenesis for Hindlimb Ischemia  567   by guest on March 12, 2014http://circ.ahajournals.org/ Downloaded from   Blood Flow Measurements There were no significant differences between treatmentgroups or time points in RBF to nonischemic limbs (Figure3). RBF in the ischemic limb exhibited an  2-fold increase inAdCMV.VEGF 121 -treated animals relative to controls as earlyas day 1 after surgery ( P  0.001). This difference remainedsignificantly higher in the AdCMV.VEGF 121  groups at allsubsequent time points. The calculated ratio of RBF in theischemic to nonischemic gastrocnemius muscles at day 1after surgery was significantly higher in AdCMV.VEGF 121 -treated animals than in controls (data not shown). By week 1,in AdCMV.VEGF 121 -treated animals, the ratio reached 1, thusindicating a complete restoration of tissue perfusion. Contrast Angiography Representative postmortem angiograms obtained at day 1 aftersurgery are shown in Figure 4. At 24 hours after femoral arteryremoval, there was an increase in the number of vessels inAdCMV.VEGF 121 -treated animals compared with controls. Inthe saline group, there was no visible collateral developmentin the thigh. In contrast, in the AdCMV.VEGF 121  (10 8 pfu/mL)group, a network of newly formed vessels developed, sproutingmainly from the internal iliac artery toward the medial thigh. TheresultingangioscorewassignificantlyhigherforAdCMV.VEGF 121 -treated animals, showing a 4-fold increase in the number of vesselscompared with animals that received saline (AdCMV.VEGF 121  50.58  1.48, saline  11.83  1.90,  P  0.0001). Animalstreated with AdCMV.Null also had a significantly higherangioscore than the saline group (Null  29.08  4.22, P  0.05), yet lower than the AdCMV.VEGF 121 -treated group( P  0.001).The qualitative angiographic assessment showed that theincreased vasculature in AdCMV.VEGF 121 -treated animalswas functional, reestablishing flow to the more distal arteriesin the leg (5 of 5 animals). Among animals that receivedAdCMV.Null, we documented distal arterial filling in theischemic leg in 2 of 5 animals, whereas none of the animalsin the saline group exhibited similar findings. Statistically,the AdCMV.VEGF 121  group differed significantly from thesaline ( P  0.002) and AdCMV.Null ( P  0.05) groups, Figure 3.  RBF to gastrocnemius muscles as a function of time after surgery. There was no difference in RBF to ischemic limb betweencontrols. Both concentrations of AdCMV.VEGF 121  were equally effective in increasing blood flow to ischemic gastrocnemius muscle asearly as day 1 after surgery. Each time point represents average of results in 4 different rabbits for each condition. * P  0.0001,† P  0.001, and ‡ P  0.01 vs controls. Figure 4.  Representative postmortemangiograms of ischemic limbs of rabbitstreated with either saline (A) or AdCMV.VEGF 121  (B) obtained at day 1after surgery. AdCMV.VEGF 121 -treatedanimals show extensive collateral arteryformation in contrast to saline-injectedanimals. Note that in AdCMV.VEGF 121 -treated animals, distal arterial filling ofischemic limb (arrow) was restored,whereas in saline group, there was noangiographic evidence of distal perfusion.  568 Circulation  August 1, 2000  by guest on March 12, 2014http://circ.ahajournals.org/ Downloaded from 

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