1. Introduction

2. Pathology of advanced PAD

3. Clinical outcomes of gene

therapy

4. Understanding the differences

between HGF and other

growth factors

5. Conclusion

6. Expert opinion

Review

Gene therapy in peripheral arterydiseaseFumihiro Sanada, Yoshiaki Taniyama*, Yasuhiro Kanbara, Rei Otsu,Yuka Ikeda-Iwabu, Miguel Carracedo, Hiromi Rakugi & Ryuichi Morishita†

†,*Osaka University Graduate School of Medicine, Department of Clinical Gene Therapy, Osaka,

Japan

Introduction: Despite the remarkable progress of medicine and endovascular

procedures for revascularization, patients with critical limb ischemia (CLI)

remain at high risk for amputation and often have a low quality of life due to

pain and ulcers in the ischemic leg. Thus, a novel strategy for generating new

blood vessels in CLI patientswithout treatment options is vital. Pre-clinical stud-

ies and Phase I clinical trials usingVEGF and fibroblast growth factor (FGF) dem-

onstrated promising results; however, more rigorous Phase II and III clinical

trials failed to demonstrate benefits for CLI patients. Recently, twomulticenter,

double-blind, placebo-controlled clinical trials in Japan (Phase III) and the USA

(Phase II) showed the benefits of hepatocyte growth factor (HGF) gene therapy

for CLI patients. Although the number of patients included in these trials was

relatively small, these results imply a distinct beneficial function for HGF over

other angiogenic growth factors in a clinical setting.

Areas covered: In this review, data from Phase I--III clinical trials of gene ther-

apy for patients with peripheral artery disease (PAD) are examined. In addi-

tion, the potential mechanisms behind the success or failure of clinical trials

are discussed.

Expert opinion: ComparedwithVEGFandFGF,HGFhasauniquemoleculareffect

on inflammation, fibrosis and cell senescence under pathological conditions.

These features may explain the clinical benefits of HGF in PAD patients.

Keywords: angiogenesis, fibroblast growth factor, gene therapy, hepatocyte growth factor,

peripheral artery disease, VEGF

Expert Opin. Biol. Ther. (2015) 15(3):381-390

1. Introduction

Despite recent therapeutic advances in preventive medicine, peripheral artery dis-ease (PAD) remains a significant global health burden that affects ~ 8.5 millionAmericans and 1.0 million Japanese [1-4]. The number of patients with PADincreases with age: 6% of individuals aged 50 -- 60 years and 10 -- 20% of thoseaged > 70 years [5,6]. The prevalence of PAD appears to be increased in developedcountries. Critical limb ischemia (CLI) is a common and devastating manifestationof PAD. The diagnosis is established when patients present with ischemic rest pain,ulcerations, or gangrene of the leg associated with evidence of reduced arterial bloodflow to the foot. The prevalence of CLI in USA is 1.3%. Within the first year ofillness, 30% suffer a major amputation (MA), 25% will die, and 20% endurewith unresolved pain or tissue loss [7]. Atherosclerosis is the principal pathologicaldisorder responsible for both CLI and coronary artery disease (CAD); thus, CLI fre-quently coincides with CAD [8-10]. Therefore, patients with CLI remain at high riskfor cardiovascular-related death. Systemic treatments, such as antiplatelet drugs,cholesterol-lowering drugs, and inhibitors of the renin-angiotensin system, havebeen widely used for the treatment of CLI. However, none of these agents currentlyimprove perfusion to the lower extremities in patients with CLI. Surgical bypass and

10.1517/14712598.2015.1007039 © 2015 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 381All rights reserved: reproduction in whole or in part not permitted

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catheter intervention can be used in PAD patients who havesimple stenosis or occlusion of large arteries. However, CLIpatients are often out of indication for revascularization dueto multiple diffuse-stenosis or calcification of the artery [11,12].Undoubtedly a novel strategy for generating new blood vesselsin CLI patients with no treatment options is indispensable. Asa potential therapy for the revascularization of ischemic tissues,therapeutic angiogenesis has garnered significant attentionover the past 20 years [13].Therapeutic angiogenesis aims at treating ischemic disease

by generating new blood vessels, and it relies on delivery ofexogenous factors or cells to stimulate neovascularization.Cell therapy using stem/progenitor cells remains at a primitivestage and is currently undergoing large clinical trials, but thistechnique shows great promise [14]. In contrast, gene therapyfor PAD patients including CLI has been validated in largetrials. The preclinical work using VEGF [15] and fibroblastgrowth factor (FGF) [16] provided encouraging data; however,no clear benefit was identified in large trials [17,18]. Recently, amulti-center, double-blind, placebo-controlled Phase III clini-cal trial in Japan and a Phase II clinical trial in the USA usingthe hepatocyte growth factor (HGF) gene therapy for CLIwere successful. Although the number of patients involved inthe trials was relatively small, in the Phase III clinical trial inJapan, a significant improvement in primary endpoints(improvement of rest pain in patients without ulcers or thereduction of ulcer size in patients with ulcers) and in thePhase II clinical trial in the USA, an increase in transcutaneouspartial oxygen pressure (TcPO2) when compared with the pla-cebo control was reported [19,20]. These results suggest thatHGF has a distinct beneficial function over other angiogenicgrowth factors in pathological conditions.This review summarizes the outcomes of clinical trials involv-

ing gene therapy for PAD andCLI patients. Subsequently, basic

aspects of angiogenic growth factors thatmay explain the successor failure of these clinical trials are discussed.

2. Pathology of advanced PAD

Successful angiogenesis using gene therapy for CLI patientsoffers relief from ischemic pain and ulcers, reduces mortalityand amputation risk, and affords a better quality of life(QOL). Understanding of CLI pathology is a prerequisite fordeveloping better therapies. CLI is the most severe form ofPAD and occurs when arterial blood flow is severely restrictedand inadequate perfusion of capillary beds leads to a loss of tis-sue viability. Compensatory mechanisms, such as capillarysprouting, typically alleviate the effects of blood flow depriva-tion.However, thesemechanisms are exhausted inCLI patients.Moreover, skeletal muscle capillary density is decreased in PADdue to a reduction in muscle use and cardiovascular risk factors,such as age, diabetes, and hypertension [21-23]. Inadequate perfu-sion of the skeletal muscle and surrounding tissues causes endo-thelial dysfunction, chronic inflammation [24,25], and muscledamage [26,27]. Inflammatory cytokines and aging acceleratethe premature senescence of endothelial cells (ECs) and stem/progenitor cells [28-33] and tissue fibrosis, which prevents oxygendiffusion and stem/progenitor cells migration toward the tissueregions needing repair. These changes can produce rest pain,chronic non-healing wounds, and gangrene [34]. Consideringthe several potential complications in CLI patients, preciseexaminations frompreclinical studies are needed to develop suc-cessful clinical trials based on angiogenic treatments.

3. Clinical outcomes of gene therapy

The administration of growth factors into ischemic tissueincreases the concentration of local angiogenic factors andaims to induce EC proliferation and migration and formationof new blood vessels in the ischemic leg. Initial preclinicalstudies demonstrated promising results in rodents [35,36].Thus, this treatment is expected to augment the functionand symptoms of patients with CLI. Initially, interest in uti-lizing angiogenic growth factors for CLI patients focused onVEGF and FGF. Recently, the therapeutic potential ofHGF was revealed (Table 1).

3.1 VEGFThe VEGF family regulates vascular growth, permeability,and angiogenesis under both pathological and physiologicalconditions [37,38]. The VEGF family consists of VEGF-Athrough VEGF-E, and VEGF-A and B have isoforms(e.g., VEGF121, VEGF165). Among the VEGF-A isoforms,VEGF165 is the most abundant and best characterized [39].Plasmid- and adenovirus vector-mediated VEGF over-expression in ischemic tissue significantly ameliorated thetissue perfusion and oxygenation accompanied by neovascula-rization in a rodent hind limb ischemia model [40,41]. Theoverexpression of VEGF165 stimulates resident ECs and the

Article highlights.

. In preclinical studies, gene therapy with the angiogenicgrowth factors VEGF, fibroblast growth factor andhepatocyte growth factor (HGF) demonstratedremarkable effects for the treatment of peripheral arterydisease. However, the data obtained from large clinicaltrials are controversial. The discrepancy may be causedby variations in patient selection criteria andassessment methods.

. Delivery of angiogenic factors targets ischemic tissuesthat are intrinsically different from normal tissues. Forinstance, critical limb ischemia (CLI) conditions limitangiogenesis potential because of endothelial celldamage and tissue fibrosis. Hence, a strategy thatdiminishes the pathology of CLI might be required forsuccessful therapeutic angiogenesis.

. Among the angiogenic growth factors, HGF has uniqueanti-inflammation, anti-fibrosis, and anti-senescenceproperties related to Ang II, ET-1 and TGF-b.

This box summarizes key points contained in the article.

F. Sanada et al.

382 Expert Opin. Biol. Ther. (2015) 15(3)

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recruitment of endothelial cell progenitor cells (EPCs) withsubsequent neovascularization [42,43]. Unlike preclinicalexperiments, the VEGF gene therapy for PAD patientsresulted in conflicting findings. In Phase I clinical trials,naked plasmid VEGF165 gene therapy was found to be safeand feasible, and it significantly promoted collateral vessels inthe ischemic tissue of CLI patients [15]. In this study, a totaldose of 4000 µg of naked plasmid DNA encoding the165-amino-acid isoform of human VEGF (phVEGF165) wasinjected twice directly into the muscles of the ischemic limb.Twelve weeks after the first administration of phVEGF165,newly formed vessels were evaluated by angiography andimmunohistochemical analysis. Moreover, Makinen et al.compared intravascular delivery of Ad-VEGF165 orphVEGF165 in patients with PAD manifesting as IC andCLI. Fifty-four patients who underwent percutaneous translu-minal angioplasty were randomized to Ad-VEGF165,phVEGF165, or placebo administration. Both VEGF165 treat-ment demonstrated to be safe and significantly increasedcapillary density when compared with the placebo controlgroup [44]. Subsequently, the efficacy of VEGF121 as an adeno-virus construct has been tested in the randomized, double-blind, placebo-controlled study encompassing a total of105 PAD patients. Patients with unilateral, exercise-limitingclaudication were randomized to receive intramuscularinjections of low-dose (4 � 109 particle units), high-dose(4� 1010 particle units) Ad-VEGF121, and placebo. The intra-muscular injection of the Ad-VEGF121 isoform failed to dem-onstrate an efficacy using the primary endpoints (change inpeak walking time) or the second endpoints (ankle-brachialindex [ABI] and QOL) after 12 or 26 weeks [45]. Additionally,no Phase III clinical trial utilizing VEGF gene transfer has dem-onstrated benefits. Notably, 60% of patients developedmoderate to severe edema in a clinical trial involving VEGF.Dose-limiting pro-inflammatory side effects of VEGF, suchas vascular permeability, leukocyte adhesion, and up-regulation of adhesion molecule expression, were reported inpreclinical studies [46-49]. New strategies, such as slow-releaselow-dose VEGF therapy with new delivery systems [50] or com-binations of VEGF with other growth factors (e.g., with HGFand angiopoietin-1), have been developed to promote neovas-cularization while limiting VEGF-induced edema and inflam-mation [51,52]. These new strategies may allow the researcher toinject a sufficient dose of VEGF for new collateral formationover the ischemic region. The differences of VEGF isoformsalso deserve considering. As VEGF121 lacks the heparin-binding domain that is required for adhesion of VEGF toextra-cellular matrix proteins, this characteristic may cause ashort tissue half-life and induce only the initial step of angio-genesis. On the other hand, VEGF165 has longer tissue reten-tion than VEGF121 and may permit better angiogenesis [53].

3.2 Fibroblast growth factorFGF is another potent angiogenic growth factor that has beenwidely studied [54]. The FGF family consists of at leastT

able

1.Humanclinicaltrials

ofangiogenic

growth

factors

forpatients

withPAD.

Trials

Vectorandpromoter

Delivery

route

Phase

Population

No

Outcomes

Baumgartneretal.(1998)[15]

phVEGF165/M

IEhCMV

Intra-m

uscular

ICLI

9Tolerated

Makinenetal.(2002)[44]

phVEGF165/M

IEhCMV

AdVEGF165/M

IEhCMV

Intra-arterial

IIPADsuitable

forPTA

54

Tolerated,increase

vascularity

RAVE(2003)[45]

AdVEGF121/M

IEhCMV

Intra-m

uscular

IIPAD,exercise-lim

itingIC

105

Noim

provementofexercise

perform

ance

orQOL

Groningen(2006)[17]

phVEGF165/notreported

Intra-m

uscular

IIDiabetics,CLI

54

Noreductionin

amputationrate

Comerota

etal.(2002)[16]

phFG

F-1/M

IEhCMV

Intra-m

uscular

ICLI

107

Tolerated

TALISMAN

(2008)[57]

phFG

F-1/M

IEhCMV

Intra-m

uscular

IICLI

125

Reductionin

amputationrate

TAMARIS

(2011)[18]

phFG

F-1/M

IEhCMV

Intra-m

uscular

IIICLI

525

Noim

provementofQOLorABI,No

reductionin

amputationrate

ordeath

Morishitaetal.(2011)[62]

phHGF/MIEhCMV

Intra-m

uscular

I/IIa

CLI,ASO,Burger,

22

Tolerated

Makinoetal.(2012)[63]

phHGF/MIEhCMV

Intra-m

uscular

I/IIa

CLI,ASO,Burger,

22

ImprovementofABI,reductionin

rest

pain

andulcersize

upto

2years

HGF-STAT(2008)[19]

phHGF/MIEhCMV

Intra-m

uscular

IICLI

104

Improvementin

transcutaneouspartial

oxygenpressure

2TREAT-HGF(2010)[20]

phHGF/MIEhCMV

Intra-m

uscular

IIICLI

40

Improvementin

rest

pain

andABI,

reductionin

ulcersize

ABI:Aankle-brachialindex;CLI:Criticallim

bischemia;HGF:Hepatocyte

growth

factor;MIEhCMV:Majorim

mediate-earlyenhancer/promoterfrom

humancytomegalovirus;PAD:Peripheralartery

disease;QOL:Qualityoflife.

Gene therapy in peripheral artery disease

Expert Opin. Biol. Ther. (2015) 15(3) 383

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23 members that bind to several spliced isoforms of the FGFreceptors, which are expressed on ECs, vascular smooth mus-cle cells, and EPCs. The activation of FGF receptors promotesthe proliferation and differentiation of the respective celltypes [55]. Among them, FGF-1 (acidic), FGF-2 (basic), andFGF-4 are highly angiogenic and facilitate the sprouting ofcapillaries from vessels in the ischemic tissue. A naked DNAplasmid vector (non-viral FGF vector; NV1FGF) carryinghuman FGF-1 was used as a gene therapy in preclinical andclinical studies. Animal studies showed the potential ofNV1FGF to induce a functional vascular network in an ische-mic leg [56]. A Phase I clinical trial was conducted byComerota et al. Fifty-one patients with unreconstructiblePAD with rest pain or tissue necrosis underwent treatmentwith intramuscular NV1FGF. Increasing single (500, 1000,2000, 4000, 8000, and 16,000 mg) and repeated (2 � 500,2 � 1000, 2 � 2000, 2 � 4000, and 2 � 8000 mg) dosesof NV1FGF were injected into the ischemic muscle.NV1FGF gene transfer was well tolerated and, in spite ofthe absence of a control group, it was suggested that treatmentimproved pain, ulcer healing, increased TcPO2, and ABI.Moreover, 33% of patients showed evidence of new bloodvessel formation at the end of the study [16]. A subsequentPhase II clinical trial (TALISMAN) also demonstratedpromising results. In this double-blind, randomized,placebo-controlled multinational study, 125 patients inwhom revascularization was not considered to be a suitableoption, presenting with non-healing ulcers, were randomizedto receive eight intramuscular injections of placebo or2.5 ml of NV1FGF at 0.2 mg/ml on days 1, 15, 30, and45 (total 16 mg: 4 � 4 mg). The primary end point wasoccurrence of complete healing of at least one ulcer in thetreated limb at week 25. Secondary end points includedABI, amputation, and death. There were 107 patients eligiblefor evaluation. Improvements in ulcer healing were similar foruse of NV1FGF (19.6%) and placebo (14.3%; p = 0.514).However, the use of NV1FGF significantly reduced (bynearly two fold) the risk of MAs or death, 51.8% in placeboand 27.4% in treatment group (hazard ratio [HR] 0.435;p = 0.009) [57]. Based on the strikingly positive results of thePhase II clinical trial, a Phase III randomized clinical trial(TAMARIS) was conducted [18]. In this Phase III trial,525 patients with CLI unsuitable for revascularization wereenrolled from 171 sites in 30 countries. All had ischaemiculcer in legs or minor skin gangrene and met hemodynamiccriteria (ankle pressure < 70 mm Hg or a toe pressure< 50 mm Hg, or both, or a transcutaneous oxygen pressure< 30 mm Hg on the treated leg). Patients were randomlyassigned to either eight intra-muscular injections of NV1FGFat 0·2 mg/ml on days 1, 15, 29, and 43 or matching placebo.The primary endpoint was MA or death at 1 year. Unfortu-nately, the primary endpoint did not differ between treatmentgroups, with MA or death in 86 patients (33%) in the placebogroup, and 96 (36%) in the NV1FGF group (HR 1.11;p = 0·48). No benefit of NV1FGF was detected in the

secondary endpoints (minor amputation, skin lesion status,pain index, QOL, and ABI/toe-brachial pressure index).Although, the baseline characteristics were comparablebetween TAMARIS and other Phase II clinical trials, theamputation and mortality rate of the control group in theTAMARIS trial was ~ 30%, whereas this rate was ~ 50% inother Phase II clinical trials. This endpoint characteristic ofthe placebo group in the TAMARIS trial might explain thelack of treatment benefits. Additionally, FGF gene therapyhas been associated with hypertension and membranousnephropathy, which must be carefully considered when deter-mining the dose and duration of FGF administration [58].Recently, the result of a Phase I/IIa open-label four dose-escalation clinical study using a new gene transfer vector basedon a nontransmissible recombinant Sendai virus (rSeV)expressing the human FGF-2 gene (rSeV/dF-hFGF2) inpatients with PAD has been presented [59]. The safety, tolera-bility, and possible therapeutic efficacy (significant and con-tinuous improvements in absolute claudication distance andrest pain) of a single intramuscular administration of rSeV/dF-hFGF2 were observed over a 6-month follow-up. Asonly 12 patients were included in this trial, larger pivotal stud-ies are warranted as a next step.

3.3 Hepatocyte growth factorAnother candidate angiogenic growth factor is HGF. HGFwas initially discovered as a potent mitogen for hepato-cytes [60]. Subsequently, its angiogenic potential was reported.The stimulation of the HGF receptor c-Met induces prolifer-ation of ECs and EPCs and migration of ECs and SMCs.Compared with VEGF and bFGF, HGF can induce angio-genesis without the stimulation of vascular senescence,inflammation caused by NFkB-induced IL-1 and monocytechemotactic protein-1, or vascular permeability caused byincreased expression of aquaporin 1 [28,61]. Based on thesepre-clinical findings, a human clinical trial (Phase I/IIa) usingintramuscular injections of naked human HGF plasmid wasconducted [62]. Twenty-two patients with CLI were treatedwith two injections of either 2 or 4 mg of naked HGF plas-mid. No edema or serious adverse events were detected. Theadministration of HGF plasmid significantly improved ABI,the size of ischemic ulcers, and the visual analog scale score2 months after treatment. The long-term follow-up of thisstudy was recently reported [63]. An ABI > 0.1 (11 of14 patients), reduction in rest pain (9 of 9 patients), anddecrease in the size of ulcers (9 of 10 patients) was observedat the 2-year follow-up without severe complications oradverse effects. Powell et al. completed another double-blindplacebo-controlled study with an HGF plasmid in theUSA [19]. TcPO2 was increased at 6 months in the high-dose group (4.0 mg at days 0, 14 and 28) when comparedwith the placebo, low-dose (0.4 mg at days 0, 14 and 28),and middle-dose (4.0 mg at days 0 and 28) groups. No differ-ence in the ABI, toe-brachial pressure index, wound healing,or incidence of MA between groups was observed.

F. Sanada et al.

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Shigematsu et al. completed a randomized, double-blind,placebo-controlled clinical trial of HGF plasmids in CLIpatients (Phase III) [20]. Forty-four patients were recruited,and a significant difference in the primary endpoint (improve-ment of rest pain and ulcer size) and improvement of QOL inthe HGF-treated group was noted. Although this trial failedto demonstrate an improvement of ABI or amputation rate,there were no major safety problems. Following these favor-able outcomes in HGF-treated patients, a global multicenterPhase III clinical trial aimed at recruiting > 500 CLI patientshas begun. Although the clinical trials using HGF gene ther-apy in PAD patients are limited, at least three randomizedplacebo-controlled clinical trials using naked human HGFplasmid DNA have demonstrated beneficial treatmenteffects [19,20,62]. Because high levels of HGF and/or cMETexpression has been reported in a variety of human cancers,including breast, lung, and GI malignancies, it requires specialattention to tumor growth with HGF gene therapy as a poten-tial side effect.

A number of clinical trials using gene therapy for CLIpatients have been performed. The results obtained from pre-clinical studies and the results from Phase III clinical trials ofVEGF, FGF, and HGF gene therapy suggest that these threeangiogenic factors have distinct molecular mechanisms thatmay predict their successful use for therapeutic angiogenesis.

4. Understanding the differences betweenHGF and other growth factors

Originally HGF was isolated from the plasma of patients withfulminate hepatic failure [60]. Currently, several roles for theHGF/c-Met system in pathological conditions have beenrevealed, including cell survival, differentiation and prolifera-tion, anti-inflammation, and anti-fibrosis [64-66]. Thus, HGFis now appreciated as a key growth factor for the attenuationof both acute and chronic disease progression in the heart [67],kidney [64], liver [68], and vascular repair [28,30]. Among thebeneficial functions of HGF, VEGF and FGF do not exertanti-inflammatory and anti-fibrotic actions. Inflammation isessential for the initiation and progression of a wide range ofchronic diseases, including PAD [69-72]. At the cellular level,the inflammatory cytokines IL-6 and IL-8 have been associ-ated with cellular senescence [73-76]. In turn, the senescent cellssecret multiple inflammatory cytokines, which drives thesenescence-cytokine loop. Thus, the relationship betweeninflammation and PAD is evident. Nonetheless, the majorityof preclinical animal studies of PAD have been performedin the absence of inflammation caused by the vascular riskfactors present in actual cases of PAD.

To explore the possible explanation for the success and fail-ure of the HGF and VEGF clinical trials, we compared theangiogenesis ability of HGF and VEGF under Ang II stimu-lation. Ang II is secreted by inflammatory cells and promotescell senescence. Interestingly, HGF, but not VEGF, attenu-ated Ang II-induced senescence of ECs and EPCs by a

reduction in oxidative stress through the inhibition of thePIP3/rac1 pathway [30]. HGF and its receptor c-Met down-regulate EGFR in a ligand-dependent manner. Thedegradation of EGFR by HGF occurs through the ubiquitinproteasome system [28]. Importantly, this system operateswhen the cells are stimulated by lipopolysaccharide, ET-1,and TGF-b1. These cytokines also trans-activate EGFR-related reactive oxygen species production. These results implythat the mechanism of ligand-dependent EGFR down-regulation by the HGF/cMet system contributes to anti-inflammatory and anti-oxidant actions in atherosclerosis [28,77].Recently, Kaga et al. documented that FGF, but not HGF,activated the inflammation-related transcription factorNFkB and its downstream inflammation-associated cytokinesin vascular smooth muscle cells, resulting in an increase in vas-cular permeability in a rat paper disc model [78]. Moreover, theexpression level of VEGF increased after neointimal injury andrecruited monocyte-lineage cells; in contrast, HGF decreasedthe recruitment of cells after injury [79,80]. Importantly, HGFand VEGF synergistically induce EC proliferation, chemotac-tic responses and neovascularization [81,82]. HGF also canreduce VEGF-induced leukocyte adhesion and adhesion mol-ecule expression by suppressing VEGF-induced NFkB signal-ing [83]. Thus, HGF exerts its angiogenic property whileinhibiting inflammation, edema, and cellular senescence.

HGF has an anti-fibrosis property, whereas VEGF andFGF induce tissue fibrosis [84,85]. We previously demon-strated that the up-regulation of HGF significantly decreasesthe fibrotic tissue area following acute myocardial infarc-tion [66]. Our recent work demonstrated that HGF signifi-cantly attenuates the transition of epithelial cells intomesenchymal cells (EMT), which is considered to beinvolved in the perivascular fibrosis of the heart [86] and kid-ney [64]. The diminution of EMT and subsequent tissuefibrosis would serve to minimize impediments to tissueregeneration. It is conceivable that intramuscular fibrosisprevents resident stem/progenitor cell migration. Engraft-ment of circulating stem/progenitor cells and oxygen diffu-sion would be limited by perivascular fibrosis and result inimpairment of tissue regeneration and oxygenation. Locallevels of HGF are decreased in an ischemic leg and intramus-cular and perivascular fibrosis cause disease progression [87];therefore, the administration of HGF using gene therapycan significantly influence organ regeneration. Fibrosiscan influence the progression of heart failure; however,HGF stimulated resident cardiac stem cell migration andimproved the cardiac function of infarcted mice hearts [88].Thus, the properties of HGF, VEGF, and FGF likelydifferentially influence angiogenesis under pathological con-ditions. We need to acknowledge the limitations of preclini-cal studies and the complexities of clinical settings;furthermore, differences in the dose and duration of genetherapy, vectors, timing of vector administration, andendpoints should also be considered while interpretingclinical trial results.

Gene therapy in peripheral artery disease

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5. Conclusion

Despite significant advances in preventive medicine, the num-ber of patients with PAD is increasing with the aging worldpopulation [89]. Due to the limitations of surgical and endo-vascular approaches, there is a pressing unmet clinical needfor angiogenic treatments for the advanced stages of PAD.Clinicians and scientists have explored therapeutic angiogene-sis using gene therapy for more than a decade. The dramaticeffects of gene therapy achieved with a single angiogenicgrowth factor were demonstrated in several animal studies;however, these findings were not fully translated into clinicalpractice. A comprehensive understanding of the biology ofneovascularization under pathological conditions can providecritical information for successful future trials of therapeuticangiogenesis. Although it is challenging to translate basicresearch to the clinical setting, HGF has a unique angiogenicpotential as a treatment for complex conditions in advancedPAD patients.

6. Expert opinion

Despite the remarkable benefits of VEGF and FGF gene ther-apy for PAD observed in preclinical studies, no benefits werefound in large trials. The pathological conditions in CLImay confound the use of a single gene therapy for angiogene-sis. Recently, HGF showed potential as a treatment for CLI inrandomized placebo-control Phase III clinical trials. HGF hasangiogenic potential and unique anti-inflammation and anti-fibrosis functions; thus, it could be used to treat the pathologyof CLI and induce angiogenesis. Although differences in thedose and duration of gene therapy and vectors can affect clin-ical outcomes, the results obtained from clinical trials mightoffer ways to improve gene therapy-induced angiogenesis.

6.1 Limitation of clinical data and future perspectivesIn this review, we discussed the difference in angiogenicpotential for HGF, VEGF, and FGF under pathological con-ditions. The interpretation of the clinical data obtained fromgene therapy for PAD patients are limitations because theoptimal dose, duration and timing of gene therapy have yetto be determined. In physiological conditions, it takes weeksor months to for newly formed vasculature to mature [90,91].

However, it is uncertain whether a persistent angiogenic stim-ulus is needed for neovascularization in an ischemic leg in theclinical setting. Thus, a more robust exploration of dose andduration strategies is needed to optimize gene therapy forPAD. Delivery route and vector selection should also be opti-mized. Other than intramuscular gene administration, newmethodology, such as ultrasound--mediated and intravenousretrograde gene delivery [92,93], can be tested in PAD patientswith main artery occlusion. These optimizations must bedemonstrated to provide benefits for patients with PAD.Importantly, angiogenesis is a complex process requiring thecoordinated interplay of several growth factors, various celltypes and the extracellular matrix. Use of a single growth fac-tor is likely insufficient to generate new vessels, which couldexplain the failure of several trials involving this method.

As the population ages, the number of patients with PADand coronary artery disease will continue to increase dramati-cally. Therefore, therapeutic angiogenesis, including genetherapy and stem cell therapy, are needed to lessen the burdenof ischemic disease and produce a better QOL. Clinical/preclinical studies with proper interpretations might offeradvances in therapeutic development.

Acknowledgments

We thank the members of the Department of Clinical GeneTherapy at Osaka University Graduate School of Medicinefor their helpful discussion and technical support. We alsothank the American Journal Expert service for languageediting manuscript.

Declaration of interest

R Morishita received honoraria, consulting fees and fundsfrom Novartis, Takeda, Shionogi, Astellas, Boehringer Ingel-heim, Daiichi-Sankyo and Pfizer. The authors have no otherrelevant affiliations or financial involvement with any organi-zation or entity with a financial interest in or financial conflictwith the subject matter or materials discussed in the manu-script. This includes employment, consultancies, honoraria,stock ownership or options, expert testimony, grants orpatents received or pending, or royalties.

F. Sanada et al.

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AffiliationFumihiro Sanada1, Yoshiaki Taniyama*2,3,

Yasuhiro Kanbara1, Rei Otsu1,

Yuka Ikeda-Iwabu1, Miguel Carracedo1,

Hiromi Rakugi2 &

Ryuichi Morishita†4 MD PhD†,*Authors for correspondence1Osaka University Graduate School of Medicine,

Department of Clinical Gene Therapy, Suita,

Osaka 565-0871, Japan2Osaka University Graduate School of Medicine,

Department of Geriatric Medicine and

Nephrology, Suita, Osaka 565-0871, Japan3Associate Professor,

Osaka University Graduate School of Medicine,

Department of Clinical Gene Therapy,

2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan

Tel: +81 6 6879 3406;

Fax: +81 6 6879 3409;

E-mail: taniyama@cgt.med.osaka-u.ac.jp4Professor,

Osaka University Graduate School of Medicine,

Department of Clinical Gene Therapy, Suita,

Osaka 565-0871, Japan

E-mail: morishit@cgt.med.osaka-u.ac.jp

F. Sanada et al.

390 Expert Opin. Biol. Ther. (2015) 15(3)

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