Vascular Pharmacology 44 (2006) 395–405www.elsevier.com/locate/vph

Review

Gene therapy of the ischemic lower limb — Therapeutic angiogenesis

Vladimir Bobek a,b,⁎, Oliver Taltynov c, Daniela Pinterova a, Katarina Kolostova a

a Third Faculty of Medicine, Charles University Prague, Department of Tumor Biology, Czech Republicb Medical Academy Wroclaw, Thoracic Surgery, Poland

c Institute of Experimental Medicine Prague, Department of Cell Ultrastructure and Molecular Biology, Czech Republic

Received 1 December 2005; accepted 1 March 2006

Abstract

The limitations of surgical revascularisation and pharmacological treatment in peripheral arterial occlusive disease (PAOD) are well recognized.Therapeutic options for critical leg ischemia are consequently limited to percutaneous transluminal angioplasty (PTA) or surgical revascularisation.Unfortunately, many patients with critical leg ischemia are poor candidates for either procedure. Therapeutic angiogenesis is a novel promising tool totreat these patients. Experimental and clinical and trials of gene transfer for therapeutic angiogenesis have already shown some clinical efficacy. Thisreview is focused on gene transfer techniques in preclinical and clinical therapeutic angiogenesis, angiogenic growth factors, vectors, deliverymethods and routes. The results of clinical and experimental studies, safety and side effects of gene therapy, and the perspectives of future research arealso discussed.© 2006 Elsevier Inc. All rights reserved.

Keywords: VEGF; Therapeutic angiogenesis; Plasmid; Vector; PAOD

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3962. Factors with angiogenic and therapeutic potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396

2.1. VEGF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3962.2. VEGF isoforms and receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

3. Gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3973.1. Gene transfer methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3973.2. Gene delivery routes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398

4. Preclinical studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3994.1. Therapeutic angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3994.2. Prevention of restenosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4004.3. Prevention of graft failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400

5. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4006. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4017. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402

⁎ Corresponding author. Third Faculty of Medicine, Ruska 87, 100 34, Prague, Czech Republic.E-mail address: vbobek@centrum.cz (V. Bobek).

1537-1891/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.vph.2006.03.009

Table 1Angiogenic growth factors and angiogenesis inhibitors

Angiogenic growth factors Angiogenesis inhibitors

Angiogenin AngioarrestinAngiopoietin-1(Ang-1)

Angiostatin(plasminogen fragment)

Del-1 Antiangiogenic antithrombin IIIFibroblast growth factors:acidic (aFGF) and basic

(bFGF) Cartilage-derivedinhibitor (CDI)

Follistatin CD59 complement fragmentGranulocyte colony-stimulatingfactor (G-CSF)

Endostatin (collagenXVIII fragment)

Hepatocyte growth factor(HGF)/scatter factor (SF)

Fibronectin fragment

Interleukin-8 (IL-8) Gro-betaLeptin HeparinasesMidkine Heparin hexasaccharide

fragmentPlacental growth factor Human chorionic

gonadotropin (hCG)Platelet-derived endothelialcell growth factor (PDECGF)

Interferon alpha/beta/gamma

Platelet-derived growthfactor-BB (PDGF-BB)

Interferon inducible protein (IP-10)

Pleiotrophin (PTN) Interleukin-12Progranulin Kringle 5 (plasminogen fragment)Proliferin Metalloproteinase inhibitors

(TIMPs)Transforming growth factor-alpha(TGF-alpha)

2-Methoxyestradiol

Transforming growth factor-beta(TGF-beta)

Placental ribonuclease inhibitor

Tumor necrosis factor-alpha(TNF-alpha)

Plasminogen activator inhibitor

Vascular endothelial growthfactor (VEGF)

Platelet factor-4 (PF4)

Prolactin 16 kD fragmentProliferin-related protein (PRP)RetinoidsTetrahydrocortisol-SThrombospondin-1 (TSP-1)Transforming growth factor-beta(TGF-b)VasculostatinVasostatin (calreticulin fragment)

396 V. Bobek et al. / Vascular Pharmacology 44 (2006) 395–405

1. Introduction

Epidemiological studies report that critical ischemia of thelimb develops in approximately 500 to 1000 people per millionper year (Second European Consensus Document on chroniccritical leg ischaemia, 2004). Direct revascularisation may beunsuccessful in most of these cases due to the anatomic extentand distribution of arterial occlusive disease (PAOD) (Dormandyet al., 1989; Isner and Rosenfield, 1993). Patients with thiscondition may eventually require amputation and may developserious morbidity and mortality (Gregg, 1985; Ouriel et al.,1988). It has been estimated that 150000 patients require lower-limb amputation for critical leg ischemia in the United Statesannually (Dormandy and Thomas, 1988).

Therapeutic options for critical leg ischemia are consequentlylimited to percutaneous transluminal angioplasty (PTA) or sur-gical revascularisation. Unfortunately, many patients with crit-ical leg ischemia are poor candidates for either procedure. Nopharmacological treatment has been shown to alter the naturalhistory of critical ischemia of the leg (Isner and Rosenfield,1993).The goal of limb salvage in this group of patients hasstimulated research into alternative treatment methods, includ-ing therapeutic angiogenesis.

Angiogenesis also called neovascularization, refers to thegrowth of new blood vessels into tissue. Therapeutic angiogen-esis is a concept that had first been introduced by the Germangynaecologist Michael Höckel in 1989 (Willard et al., 1994).The idea was to induce capillary growth in order to improveregional tissue perfusion (incl. oxygen supply) and to improvetissue viability following surgery. In contrast to angiogenesis,arteriogenesis is not dependent on hypoxia/ischemia. Arterio-genesis is a process of transformation of a small arteriole intomuch larger conductance artery. The presence of arteriogenesisis genetically determined, because pre-existent arterioles are es-sentials (Schaper and Scholz, 2003).

This review will focus on therapeutic angiogenesis based ongene transfer techniques for the treatment of limb ischemia.

2. Factors with angiogenic and therapeutic potential

Vascular endothelial growth factor A (VEGF-A), Fibroblastgrowth factor (FGF), Angiopoetin 1 (Ang-1) Insulin growthfactor-I (IGF-I) and Hepatocyte growth factor (HGF) are mem-bers of a group of potent angiogenic growth factors whichtogether play critical roles in determining the structure andfunction of blood vessels in process of angiogenesis or arte-riogenesis (Table 1).

2.1. VEGF

VEGF-A is a key regulator of physiological angiogenesisduring embryogenesis, skeletal growth and reproductive func-tions. VEGF-A has also been implicated in pathological angiogen-esis associated with tumours, intraocular neovascular disordersand other conditions. A well-documented in vitro activity ofVEGF factors is the ability to promote growth of vascular en-dothelial cells (ECs) derived from arteries, veins and lymphatics

(Ferrara et al., 2003). The placental growth factor (PLGF),VEGF-B, VEGF-C, VEGF-D belong to the VEGF family, too(Maglione et al., 1991). Recent studies have shown that VEGFstimulates surfactant production (Compernolle et al., 2002) andinduces lymphangiogenesis (Matsumoto and Claesson-Welsh,2001), VEGF enhances chemotaxis and migration of vascularsmooth muscle cells (SMCs) and coordinates longitudinal bonegrowth and endochronal bone formation (Springer et al., 2000;Gerber et al., 1999). One important feature of VEGF is its che-motactic effect on circulating monocytes and other leukocytes,which are inducers of vascular growth, as sources of growthfactors and cytokines (Arras et al., 1998; Couffinhal et al.,1999). VEGF delivery to adult mice inhibits dendritic cell de-velopment (Gabrilovich et al., 1996) and increases productionof B cells (Hattori et al., 2001). VEGF is also known as a

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vascular permeability factor, based on its ability to inducevascular leakage (Dvorak et al., 1995). VEGF mRNA expres-sion is induced by exposure to low oxygen values under avariety of patophysiological circumstances (Dor et al., 2001).Hypoxia-inducible factor (HIF)-1 is a key mediator of hypoxicresponses. This transcription factor can activate a collection ofdifferent genes that are involved in angiogenesis, includingthose encoding VEGF receptors, and angiotensin-2 (Oh et al.,1999; Semenza, 2000).

2.2. VEGF isoforms and receptors

Native VEGF-A is a heparin-binding homodimeric glyco-protein of 45 kDa (Ferrara and Henzel, 1989). The properties ofnative VEGF closely correspond to those of VEGF165 (Houck etal., 1992). Variants of the VEGF isoforms with other number ofamino acids, after signal sequence cleavage, have been alsoreported, such as VEGF121, VEGF145, VEGF183, VEGF189, andVEGF206 (Tischer et al., 1991; Ferrara and Henzel, 1989;Neufeld et al., 1999). The VEGF binding sites were identified onthe cell surface of vascular ECs in vitro and in vivo. Four dif-ferent receptors are known to bind the members of the VEGFfamily: VEGFR-1, VEGFR-2, VEGFR-3 and neuropilin-1.VEGF binds to both VEGFR-1 and VEGFR-2, but the latterreceptor mediates the mitogenic and antiapoptotic signalling(Dor et al., 2002; Rissanen et al., 2001; Saaristo et al., 2002;Yoon et al., 2003; Isner et al., 2001). FGF stimulates the pro-liferation of cells of mesodermal and neuroectodermal origin,including endothelial cells, smooth muscle cells and myoblasts(Galzie et al., 1997). FGF and VEGF have synergistic effects(Rissanen et al., 2003). The FGF family has 23 known membersin vivo (Shyu et al., 1998). HGF, as a multifunctional protein,has been reported to augment collateral vessel development,angiogenesis and potentiate the VEGF effect (Aoki et al., 2000;Morishita et al., 1999; Van Belle et al., 1998). HGF has anantiapoptotic effect on the cardiomyocytes (Nakamura et al.,2000). The mitogenic effect of HGF is very important for re-generation in skeletal muscle (Rissanen et al., 2001). IGF-I playsan important role in the regeneration and growth of peripheralnerves and skeletal muscle, and has been investigated as a treat-ment for neuromusculars disorders (Cheng et al., 1996) andmuscle regeneration following injury (Menetrey et al., 2000).Treatment with exogenous IGF-I protein reduces muscle de-generation and atrophy in dystrophic mice. IGF-I is also apotential angiogenic factor. IGF-I receptors have been shown tobe present on endothelial cells of bone (Fiorelli et al., 1996),retina (Spoerri et al., 1998), and aorta (Kobayashi and Kamata,2002). IGF-I induces the expression of VEGF mRNA on retinalpigment epithelial cells (Punglia et al., 1997), osteoblasts(Akeno et al., 2002), vascular endothelial cells (Miele et al.,2000), and in a variety of tumour cells (Reinmuth et al., 2002;Wu et al., 2002; Bermont et al., 2000). IGF-I induces cellmigration and tubular formation of cultured bovine retinalendothelial cells in vitro (Castellon et al., 2002; Shigematsu etal., 1999). IGF-I also acts as a vasoactive factor by inhibitingvessel contraction, via stimulation of nitric oxide production(Walsh et al., 1996). Finally, IGF-I plasmid therapy promotes in

vivo angiogenesis (Rabinovsky and Draghia-Akli, 2004). Ang-1is essential for remodelling and stabilization of blood vessels(Asahara et al., 1998). Ang-1 hasmigratory and sprouting effectson ECs (Chae et al., 2000) and stimulates the formation ofpericytes and smooth muscle cells with. Ang-1 binds to thetyrosin kinase receptor on endothelial cells, whereas the Ang-2acts in general as antagonists of Ang-1. Ang-2 was not beenreported to augment angiogenesis. Over-expression of Ang-1has been shown to produce highly branched and numerousleakage resistant blood vessels (Suri et al., 1998).

3. Gene transfer

3.1. Gene transfer methods

Gene transfer is focused on the introduction of foreignnucleic acids into target cells in order to achieve a localized,sustained therapeutic over-expression of the chosen gene. Thesystems can be roughly divided into two major categories, viraland non-viral.

Among the variety of different approaches for non-viral genetransfer into the vascular system, the most commonly used aredirect incubation with unmodified (naked) DNA and coupling ofDNA with lipophylic/hydrophobic agents. The use of nakedDNA is simple and well tolerated by recipient organism due tothe low toxicity and low immune response compared to viralvectors. The use of naked DNA is theoretically limited by lowtransfection efficiency, which translates into a low level oftransgene expression. When injected intravenously, plasmidDNA is very rapidly degraded in the reticulo-endothelial systemand has an extremely short plasma half-life (Tomlinson, 1996).However, plasmid DNA delivered directly to tissues can inducelocal transgene expression. Although the transfection efficiencyrate is low in muscle, transgene expressions persist for severalmonths in the absence of evidence indicating plasmid replicationor integration. Delivery of a plasmid containing complementaryDNA for VEGF to muscle or the blood vessel wall has beenfollowed by local VEGF expression and by increase in VEGFcirculation levels lasting 15 days (Isner et al., 1996).

To enhance cell uptake of naked DNA many cell electro-poration methods were applied and a variety of compounds, e.g.cationic phospholipids (liposomes), have been coupled to DNA.Liposomes facilitate the transport of DNA across the cell mem-brane using cationic polymers (Simberg et al., 2001). Liposomeshave been shown to be effective in the transfer of growth factorsin animal models of angiogenesis (namely with experimentswith VEGF and HGF). Cell targeting may be achieved by con-jugating specific target proteins to the DNA/liposome complex.After conjugation, the liposome particle will preferentially enterthose cells with appropriate receptors on their surface (Remyet al., 1994; Puyal et al., 1995; Legendre and Szoka, 1993;Demeneix et al., 1994).Transmission efficiency of plasmidDNAmay be improved by the use of ultrasound, too. Ultrasoundexposure with micro-bubble echocontrast agents increasestransgene expression significantly in naked DNA transfectionby cell membrane permeabilisation. This technique of mem-brane permeabilisation or acoustic cavitation was reported to

398 V. Bobek et al. / Vascular Pharmacology 44 (2006) 395–405

increase transgene expression by approximately 300-fold, cre-ating transient small holes in the cell surface membrane throughwhich naked DNA is rapidly translocated (Lawrie et al., 2000).In a study of the luciferase plasmid transfection with use ofultrasound, the plasmid DNA transmission efficiency increasedapproximately 10-fold compared to plasmid alone in culturedhuman skeletal muscle (Taniyama et al., 2002). Because genetransfer efficiency with plasmid-based systems is usually rel-atively low the magnetic DNA nanospheres containing expres-sion plasmids encoding VEGF were administrated via an arteryinto a rabbit limb ischemia model. Gene delivery of such nano-spheres via artery under a magnetic field led to the over-expression of VEGF in situ. The capillary density and capillaryto muscle fiber ratio were doubled compared with those of thecontrol animals. The results suggest that intra-arterial VEGFgene delivery by magnetic DNA nanosphere promotes angio-genesis and arteriogenesis (Jiang et al., 2005).

Viral vectors have been created to increase the efficiency oftransfection process. The most commonly used viral vector forgene transfer is adenovirus, which can transduce both dividingand non-dividing cells. Transfection efficiency is about 1000times better with adenoviral vectors than with plasmid DNA(Teiger et al., 2001). The major limitation of adenoviral vectorsare lack of sustained expression, as the viral DNA does notintegrate into host genome; antigenicity of viral proteins andpossible toxicity at high doses. In human trials, adenoviralvectors have caused inflammatory reactions, formation of anti-bodies to adenovirus, transient fever and significant increase ofliver transaminases, but the linkage to any human malignancyhas not been proved. The promise of adenovirus vectors for genedelivery has been tempered by their lack of tissue specificity(Harris and Lemoine, 1996). Without alteration, adenovirusescannot efficiently infect ECs and SMCs. Recently, modifiedadenoviruses have been generated to bind the alternative at-tachment receptors improving the transduction efficacy (Kibbeet al., 2000; Wickham, 2000).

Others for angiogenic gene transfer used vectors includeretroviruses, lentiviruses and adenoassociated viruses (AAVs).Advantage of the AAVs vectors for gene transfer includes thetransduction of non-proliferating cells, lasting transgenic expres-sion, and reduced inflammatory response, while limitations in-volve difficulty with production and a small packaging capacity.AAVs can also efficiently transduce skeletal, muscle, myocar-dium and blood vessels (Svensson et al., 1999; Monahan andSamulski, 2000). Other viral gene transfer vectors use herpessimplex virus, baclovirus, Sendai virus. Lentiviruses can alsotraduce non-dividing cells and have shown relatively hightransduction efficiencies in the central nervous system and liver(Trono, 2000; Naldini et al., 1996). Some investigations weredesigned to determine the effects of lentiviral-delivered vascularendothelial-derived growth factor (VEGF) and angiopoietin-2(Ang-2) on collateralization in a rabbit model of hind limbischemia. Self-inactivating human immunodeficiency virus(HIV)-based vectors were constructed encoding VEGF orAng-2, co-transfected with vesicular stomatitis virus glycopro-tein (VSV G) into 293T cells, and vector supernatants (1×108 IU/ml after concentration) were harvested. Arterial col-

lateralization and systolic blood pressure increased significantlyfollowing VEGF vector administration. Development of theantibody against VSV G can be limited by repeated injections ofvector. (Conklin et al., 2005).

3.2. Gene delivery routes

The ideal delivery method should be capable of transfectingthe target tissue with no systemic exposure to the vector. Threemethods have been used for gene delivery into skeletal muscleto treat peripheral artery: catheter mediated intravascular genetransfer, direct intramuscular injection, and ex vivo genetherapy.

A human clinical trial using VEGF was started in 1994 byProfessor Isner. The initial trial used a hydrogel catheter withnaked VEGF165 plasmid. The technique involves ballooninflation, and thus the potential for vascular injury, the site ofgene transfer should be serially assessed by intravascularultrasound. The hydrogel was used as a carrier of the plasmidDNA (Morishita, 2002).

A study comparing three different catheter-based strategiesand a surgical technique has shown that catheters permitrelatively efficient adenovirus-mediated gene transfer tovascular endothelium (Willard et al., 1994). Lower extremityvascular disease is often so extensive that conventional sites forarterial puncture cannot be assessed percutaneously. Arterialsites may be diffusely diseased by atherosclerosis (Feldman etal., 1995). Even in the absence of a thickened neointima,extensive calcification at the intimal-medial interface may limitgene transfer to the vascular cells and make the vessel so brittlethat balloon inflation fractures the calcified vessels, leading tounpredictable abrupt vessel closure (Fitzgerald et al., 1992).This complication can be devastating if the involved artery isthe major donor of existing collaterals or only patent vesselsupplying the ischemic limb. Even if arterial access is possiblein such patients, it is often limited to the uppermost portion ofthe limb, 60 cm or more from sites in the distal limb whereischemia or necrosis is most profound (Takeshita and Isner,1999). On the other side intra-arterially administered vectorleads to a more extensive biodistribution than the vector injectedintramuscularly (Hiltunen et al., 2000b).

Direct gene transfer of plasmid DNA or viral vector intoischemic limb muscles is a less invasive therapeutic alternativeto arterial transfection. The efficiency of intramuscular genetransfer is augmented when the injected muscle is ischemic(Takeshita et al., 1996). Higher and less variable geneexpression could be achieved by injecting a larger rather thana smaller volume of plasmid (Wolff et al., 1991). Pre-injectionof muscles with relatively large volume of hypertonic sucrosefacilitated more uniform distribution and less variable expres-sion of delivered genes (Davis et al., 1993). From clinicalstandpoints, these findings suggest that intramuscular genetransfer represents a suitable alternative to arterial gene transferin patients with proximal obstruction of the lower extremityvasculature.

However none of the methods of gene transfer mentionedabove ensure that only the target cells are transfected.

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Introducing foreign DNA into non-target cells may causeadverse effects. Thus, more recently there has been considerableinterest in ex vivo gene transfer-harvesting cells, which are thentransfected, in vitro before being replaced (Ohara et al., 2001;Ninomiya et al., 2003). This method increases the transfectionefficiency and ensures that foreign DNA is only introduced intotarget cells. Ex vivo VEGF gene transfer to myoblasts wasperformed followed by the implantation of these cells intononischemic murine legs (Springer et al., 1998).

Also another alternative cell therapy approach to induce an-giogenesis was proposed. Small organ fragments whose geome-try allows preservation of the natural epithelial/mesenchymalinteractions and ensures appropriate diffusion of nutrients andgases to all cells were prepared. Fragments derived from thelungs are shown to behave as fairly independent units, to un-dergo a marked upregulation of angiogenic factors and to con-tinue to function for several weeks in vitro in serum-free media.When implanted into hosts, they transcribe a similar array ofangiogenic factors that specifically induce the formation of apotent vascular network. The angiogenic induction capacity ofthese fragments was also tested in a mouse and rat model of limbischemia. Such fragments, when implanted in the vicinity of theischaemic area, induce an angiogenic response which can rescuethe ischaemia-induced damage. The approach presented differsfrom single factor application, gene therapy and other celltherapy methods in that it exploits the complex behaviour ofautologous cells in their near to normal environment in order toachieve secretion of a whole range of angiogenic stimuli con-tinuously and in an apparently coordinated fashion (Hasson etal., 2005).

Bacteria, which produce angiogenic factors, provide a newmodality for experimental angiogenesis and may be also suitablefor clinical use. Escherichia coli strain BL21(DE3) was trans-formed with Bluescript vector containing the inserts with cDNAsequences coding VEGF-A isoforms (VEGF121, VEGF164,VEGF189). The expression of target genes in the T7 expressionsystemwas induced by isopropyl-beta-D-thiogalactoside (IPTG).Blood vessel formation induced by bacterial VEGF productionwas proven in vivo in mice seven days after intraperitoneal in-jection of transformed bacteria by light microscopy. The mainadvantage of the described approach lies in the enhanced reg-ulation control–bacterial expression that can be regulated posi-tively (induction by exogenous lowmolecular weight agents) andnegatively (application of antibiotics) (Celec et al., 2005).

4. Preclinical studies

Gene therapy for peripheral vascular disease focuses cur-rently on three areas: (1) therapeutic angiogenesis— stimulationof blood vessel growth, (2) preventing restenosis after balloonangioplasty or stent placement, and (3) preventing failure ofvascular grafts.

4.1. Therapeutic angiogenesis

Widely used animal models in studies of therapeutic angio-genesis are the rabbit hind-limb acute ischemia model (Pu et

al., 1993; Tsurumi et al., 1996), the mouse and rat models(Couffinhal et al., 1998; Mack et al., 1998; Takeshita et al.,1998).

In animal models, therapeutic effects have been shown byrecombinant growth factors administered intraarterially, in-travenously or intramuscularly (Takeshita et al., 1994a,b;Bauters et al., 1995; Tsurumi et al., 1996; Garcia-Martinez etal., 1999; Shyu et al., 2003). Growth factors that have beenshown to generate therapeutic angiogenesis in animal studies, asrecombinant protein or gene therapy, are listed in Table 1. Thereis an overwhelming evidence on the usefulness of VEGF, FGFin angiogenic therapy in vivo compared to others, making thesegrowth factors prime candidates for therapeutic drugs.

Several application and vector systems work in mice andrabbits, but it is more difficult to obtain equal treatment efficacyin larger animals as pig. So, low gene transfer efficiency is amajor problem in human gene therapy. This is because oflimited tissue diffusion of the gene transfer vectors and largervolumes of the transfected tissues, such as skeletal and myo-cardial muscles. Also tissue damage caused by manipulation ofsmall animals, especially in the myocardium and skeletal mus-cle can significantly increase transduction when compared tointact tissues (Wright et al., 2001; Vitadello et al., 1994). Anadditional concern is that preclinical studies have been donein healthy young animals that are able to mount an effectivetherapeutic response, whereas such a capacity may not be pres-ent in elderly patients with atherosclerotic blood vessels, dia-betes or other chronic disease processes (Ferrara and Alitalo,1999). Some investigations were designed to determine theeffects of lentiviral-delivered vascular endothelial-derivedgrowth factor (VEGF) and angiopoietin-2 (Ang-2) on collat-eralization in a rabbit model of hindlimb ischemia. Self-inac-tivating human immunodeficiency virus (HIV)-based vectorswere constructed encoding VEGF or Ang-2, co-transfected withvesicular stomatitis virus glycoprotein (VSV G) into 293T cells,and vector supernatants (1×10(8) IU/ml after concentration)were harvested. Arterial collateralization and systolic bloodpressure increased significantly following VEGF vector admin-istration. Development of antibody against VSV G can be lim-ited by repeated injections of vector. (Conklin et al., 2005).Preclinical animal studies have indicated that angiogenicgrowth factors can stimulate the development of collateral ves-sels, resulting in therapeutic angiogenesis. It is becoming clearthat trials of single angiogenic growth factors are not achievingthe results anticipated from experimental studies, and thereforeadministration of multiple agents may be necessary to optimizethe angiogenic response (Ohara et al., 2001). For example,the combination of VEGF and bFGF has synergistic effects(Ninomiya et al., 2003). The monocistronic vectors encodingVEGF165 or FGF-2 and bicistronic construct expressing bothof them were also tested in therapeutic angiogenesis. It wasshown that after 3, 13, 21, 31 and 41 days post-transfection, theplasmid DNA still persisted in tissue, more or less on the samelevel but the mRNA transcripts slowly decreased after 13 days.(Malecki et al., 2003). A combination of the Ang-1 and VEGFgene transfer has been reported to result in lager vessels (Chae etal., 2000).

400 V. Bobek et al. / Vascular Pharmacology 44 (2006) 395–405

4.2. Prevention of restenosis

Restenosis after balloon angioplasty is a multifactorial pro-cess, where the main mechanisms are excessive neointimaformation and unfavourable late remodelling (Mann, 2000).Important processes during the development of restenosis aremedial SMCs proliferation and migration, down regulated apo-ptosis, and increased formation and decreased degradation ofextracellular matrix. Most gene therapy strategies are directedtowards inhibition of SMC migration and proliferation, forma-tion of connective tissue, and undesirable growth factor effects(Yla-Herttuala and Martin, 2000). Inhibition of gene expressionprerequisite for SMC proliferation has been gained by antisensegene therapy in which the transferred DNA is designated toinhibit the expression of a specific host gene. Antisense ordecoy oligonucleotide constructs against c-myb, c-myc, cdc-2,cdk-2, ras, bcl-x, NFêB, E2F and TGF-β have decreased intimalthickening in experimental restenosis (Yla-Herttuala andMartin, 2000; Hedin and Wahlberg, 1997; Yamamoto et al.,2000). Animal models showed that catheter-mediated VEGFdelivery at the site of vascular injury after endothelial denuda-tion or stent placements accelerated reendotelialization, leadingto the inhibition of neointimal thickening, the reduction ofthrombogenicity and the restoration of endothelium-dependentrelaxation (Asahara et al., 1996; Hiltunen et al., 2000a).

Thromboresistance after PTA or stent placement can beenhanced by gene transfer of hirudin, tissue plasminogen acti-vator, cyclooxygenase, and thrombomodulin of tissue factorpathway inhibitor. Prevention or rapid dissolution of thrombusmay decrease the restenotic process (Riesbeck et al., 1998; Kuoet al., 1998; Rade et al., 1996; Waugh et al., 1999a,b; Zoldhelyiet al., 1996, 2000; Nishida et al., 1999).

4.3. Prevention of graft failure

Seeding of vein grafts with transfected ECs has been done inanimal models (Kupfer et al., 1994). In a model of cholesterol-fed rabbits it was demonstrated that an intraoperative genetherapy approach on vein grafts with antisense oligonucleotideblockage of medial SMCs proliferation prevented the acceler-ated atherosclerosis that is responsible for autologous vein graftfailure (Mann et al., 1995).

5. Clinical studies

Clinical trials of angiogenic therapy with recombinant pro-teins or genes have been aimed mainly to treat an inoperablechronic critical limb ischemia.

Angiogenic gene therapy was first used in 1994 in a patientwith stage III/IV peripheral occlusive arterial disease (PAOD). Acatheter placed in the vascular lumen was used to inject plasmidscontaining VEGF complementary DNA into arterial wall up-stream from the occlusion (Isner et al., 1996). Functional andangiographic parameters improved within 12 weeks, and spiderangiomata and edema developed unilaterally in the affectedlimb, demonstrating clearly that the treatment had a local angio-genic effect.

This initial trial used a hydrogen catheter with a nakedVEGF165 plasmid and although it appeared to be effective instimulating collateral formation in patients with PAOD, it is notideal for the many patients who lack an appropriate targetvascular lesion for catheter delivery. Thus, Professor Isner'sgroup trailed intramuscular injection of naked plasmidencoding the VEGF165 gene. Intramuscular application ofnaked DNA demonstrated clinical efficacy for the treatmentPAOD (Baumgartner et al., 1998; Isner et al., 1998).

Since this study numerous angiogenic growth factors, suchVEGF, FGF or HGF have been tested in clinical trials (Table 2).In addition to intramuscular injection of naked DNA plas-mid, adenoviral delivery and liposomal delivery of angiogenicgrowth factor have also been trailed. A recent trial using ade-novirus-encoding VEGF121 demonstrated improved endotheli-al dysfunction in response to acetylcholine or nitroglycerine(Rajagopalan et al., 2001), but there was high incidence ofedema as a side effect. There was no evidence of edema inany of the patients transfected with the human HGF gene,on the contrary in VEGF trial 60% of patients developedmoderate or severe edema in a phase I/II trial (Baumgartner etal., 1998; Isner et al., 1998). Although these results are stillpreliminary, gene therapy using HGF has potential in thetreatment of PAOD with minimal incidence of edema. VEGF-induced edema responses to oral diuretic therapy (Baumgartneret al., 2000) may be prevented by a combination therapy withangiotensin 1, which maintains endothelials integrity (Thurstonet al., 2000).

Diffusion of angiogenic factors such as VEGF in the bodycarries a risk of complication and side effects. However, safetyrecords from the angiogenic gene therapy trials indicate nomajorproblem. Many of the potential side effects apparent fromexperiments using transgenic and knockout animals, such asworsening of atherosclerosis or retinopathy, have not been de-tected in clinical trials (Grines et al., 2002; Makinen et al., 2002;Hedman et al., 2003; Laitinen et al., 2000). Incidences of cancerin patients undergoing angiogenic gene therapy has been thesame or lower than that in the general population of the same age(Grines et al., 2002; Makinen et al., 2002; Hedman et al., 2003).There is no compelling evidence that VEGF present in thebloodstream accelerates tumour growth or metastasis generation(Folkman, 1998). Treatment with VEGF or FGF has been welltolerated in the first clinical studies. In addition to VEGF-induced lower limb edema, other side effects reported fromangiogenic trials have been a transient increase in C-reactiveprotein, proteinuria, and thrombocytopenia (Baumgartner et al.,1998; Laitinen et al., 2000; Makinen et al., 2002; Rissanen et al.,2001).

The clinical trials for prevention of restenosis were carriedout. At the site of PTA, VEGF could have a vasculoprotectiveeffect with resultant prevention of restenosis. Analysis ofenrolled patients revealed a statistically significant increase invascularity distal to the gene transfer site at digital subtractionangiography (DSA) three months after the intervention in thegene therapy groups (Makinen et al., 1999). However, at thisstage of the trial no statistically significant difference wasdetected in the clinical outcome. No major gene-transfer-related

Table 2Clinical studies using gene therapy for revascularization of lower limb peripheral arterial occlusive disease (PAOD)

Investigator Disease Treatment Vector Delivery route Patients Reference

Baumgartner I et al. PAOD VEGF A 165 Naked DNA IM injection 9 (Baumgartner et al.(1998))

Isner JM et al. Burger disease VEGF A 165 Naked DNA IM injection 6 (Isner et al.(1998))

Isner JM et al. PAOD,stent restenosis

VEGF A 165 Naked DNA Hydrogel-coated balloon 28 (Isner (1998))

Isner JM et al./Vascular Genetics Inc.

PAOD VEGF C Naked DNA IM injection 28 –

Makinen K et al. PAODPost-PTA stent

VEGF A 165 Naked DNA Infusion–perfusion/channel catheter 54 (Makinen et al.(1999))

Laitinen M et al. PAOD LacZ Adenovirus Infusion–perfusion/channel catheter 10 (Laitinen et al.(1998))

Collateral Therapeutics/Schering AG

PAOD FGF Adenovirus IM injection Enrolling

Aventis PAOD FGF Plasmid IM injection EnrollingEurogene Ltd. PAOD VEGF A 165 Liposome Adventitioal delivery by biodegradable reservoir Enrolling (5)Mann MJ el al. Vein bypass E2F decoy Ex vivo delivery Oligonucleotide Enrolling (41) (Mann et al.

(1999))Kim H-J et al. PAOD VEGF A 165 Naked DNA IM injection 9 (Kim et al. (2004))Shyu K-G et al. PAOD VEGF A 165 Naked DNA IM injection 21 (Shyu et al. (1998))

401V. Bobek et al. / Vascular Pharmacology 44 (2006) 395–405

side effects or differences in basic laboratory tests were found.No marked limb edema was detected.

A randomized, controlled trial to limit intimal hyperplasiastenosis in infrainquinal vein bypass grafts by cell-cycle block-age with ex vivo gene transfer of E2F decoy was reported (Mannet al., 1999). E2F decoy oligonucleotide was delivered to graftsintraoperatively by ex vivo pressure-mediated transfection. Themean transfection efficiencywas 89%. At 12months, fewer graftocclusions, revisions or critical stenoses were documented in theE2F decoy group than in the untreated group.

An attractive target for gene therapy is lymphedema. Thera-peutic lymphangiogenesis is an area where no adequate clinicaldata is yet available, although it may be a potential treatment forsome severally affected individuals. In preclinical models oflymphedema and lymphatic vessel hypoplasia, the lymphaticvessel could be regenerated by using adenovirus or AAVs-mediated transduction of VEGF (Roberts and Palade, 1995;Maxwell and Ratcliffe, 2002). The newly generated lymphaticvessels were stable and functional. Improvement of lymphe-dema and restoration of normal tissue architecture was also ob-tained with recombinant VEGF in a rabbit model of postsurgicalsecondary lymphedema (Ku et al., 1993), and consistent resultswere published from studies using plasmid transfer (Safran andKaelin, 2003).

6. Perspectives

Early studies involving the administration of VEGF showedangiographic evidence of new vessel formation, but these ves-sels did not persist and they regressed within three months (Isneret al., 1996). So, one of the major problems encountered in theuse of VEGF is that the vessels formed are unstable and leaky(Dvorak et al., 1999). The vessels generated by VEGF are usu-ally “capillary-like” by nature, whereas those produced by FGFappear to be more mature. It has been speculated that VEGF

alonemay not be sufficient to form stable, mature vessels that arecharacterized by the recruitment of the perivascular mural cells,such as pericyties or SMCs (Ng and D'Amore, 2001).

Various growth factors such Ang-1, PLGF, TGF-β as well asVEGF are involved into the process of obtaining stable andmature vessels. (Thurston et al., 1999). Administration of sub-maximal doses of Ang-1 and VEGF in a rabbit ischemic hindlimb model led to a stronger effect on resting and maximal bloodflow and capillary formation than either of the agents alone(Chae et al., 2000).

Another approach that addresses the involvement of multiplefactors in therapeutic angiogenesis, is in the use of so-called“master switch gene” of angiogenesis, such as HIF-1α (Li et al.,2000). It is hoped that using a “master switch gene”will result inmore stable vessels, because the processes by which they areformed would resemble more closely those of normal vesseldevelopment.

The possibility of using stem cells in therapeutic angiogenesisis also of a big interest. The existence of circulating endothelialprecursor (CEP) cells in adults has been reported (Asahara et al.,1997; Shi et al., 1998). In an in vitro model of angiogenesis,normal vascular development has been shown to require thepresence of the CD45+/c-Kit+/CD34+ hematopoetic stem cells,which are similar and may be related to adult CEP cells. It hasbeen reported that CEP and similar precursor cells are able toparticipate in new vessel growth in a variety of animal models,including the rabbit ischemic hind limb model (Yamashita etal., 2000; Asahara et al., 1999). The possibility of using CEPcells, both alone and in combination with different angiogenicgrowth factors, represents a promising means of obtaining stablevessels.

Recently the effect of VEGF is not restricted to the directangiogenic effect in vivo but includes mobilization of bone-marrow-derived endothelial progenitor cells and augmentationof postnatal vasculogenesis in situ (Yoon et al., 2004).

402 V. Bobek et al. / Vascular Pharmacology 44 (2006) 395–405

There is also the possibility to transplant VEGF-expressingmesenchymal stem cells MSCs which could effectively treatacute myocardial infarction (MI) by providing enhanced cardio-protection, followed by angiogenic effects in salvaging ischemicmyocardium (Matsumoto et al., 2005)

7. Conclusion

Therapeutic angiogenesis seems to be a promising therapy forpatients with lower limb ischemia. Clinical trials of gene transferfor therapeutic angiogenesis in the treatment of ischemic limbhave shown some clinical efficacy. Future clinical studies areneeded to determine how to achieve optimal therapeutic angio-genesis.Many aspects of gene transfer, including the appropriatevector dose, the delivery route, and the growth factor combina-tion have to be proven.

Acknowledgments

The study was supported by research grant MSM0021620817 from Third Faculty of Medicine Charles Univer-sity Prague.

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