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PII S1050-1738(02)00256-6 TCM

Insights from Angiogenesis Trials Using Fibroblast Growth Factor for Advanced Arteriosclerotic Disease

Rohit Khurana and Michael Simons*

The aim of therapeutic angiogenesis in cardiovascular disease states isto improve myocardial and peripheral extremity perfusion and func-tion within ischemic regions that are not amenable to traditionalmodes of revascularization. Substantial “proof of concept,” efficacy,and safety data have emerged from numerous animal models and clin-ical trials that fibroblast growth factor (FGF), when administered byvarious delivery strategies, has a therapeutic angiogenic capacity. Thisinitial excitement has been replaced by cautious optimism in the wakeof results from larger, randomized, double-blinded placebo-controlledtrials of both FGF gene and protein administration. A greater under-standing of the profound placebo effect, careful patient selection, andimproved endpoint assessment are factors that need to be addressed inthis rapidly evolving era of molecular therapeutics.

(Trends CardiovascMed 2003;13:116–122)

© 2003, Elsevier Science Inc.

Rohit Khurana and Michael Simons are atthe Section of Cardiology and AngiogenesisResearch Center, Dartmouth Hitchcock Med-ical Center, Dartmouth Medical School, Leb-anon, New Hampshire, USA.

* Address correspondence to: MichaelSimons, MD, Section of Cardiology, Dart-mouth Hitchcock Medical Center, One MedicalCenter Drive, Lebanon, NH 03756, USA. Tel.:(

�

1) 603-650-3540; fax: (

�

1) 603-650-5171;e-mail: michael.simons@dartmouth.edu.

© 2003, Elsevier Science Inc. All rightsreserved. 1050-1738/03/$-see front matter

• FGF Biology

Fibroblast growth factors (FGFs) are afamily of 22 closely related proteins thatshare certain structural features and theability to interact with specific receptors(Ornitz and Itoh 2001). FGFs elicit diversebiologic effects on numerous cell types(fibroblasts, endothelial cells, smoothmuscle cells, and keratinocytes, amongothers). These effects encompass stimu-lation of growth, proliferation, migration,

TCM Vol. 13, No. 3, 2003

117

and differentiation and are transmittedby an elaborate FGF signaling systemthat includes four high-affinity tyrosinekinase receptors, a transmembranesyndecan-4 core protein, and likely othermembrane signaling molecules. These fourtyrosine kinase FGF receptors (FGFRs)share 55% to 72% structural homology(Johnson and Williams 1993). The struc-tural motifs that constitute the trans-membrane FGFR are three extracellularimmunoglobulin (Ig)-like domains (des-ignated IgI, IgII, and IgIII), an acidicregion between IgI and IgII, a trans-membrane domain, and an intracellulartyrosine kinase domain (for a review, seePowers et al. 2000). Alternative splicingof the IgIII domain generates anothersource of variation for FGFRs 1, 2, and3, which are designated IgIIIa, IgIIIb,and IgIIIc. The FGFR4 gene is unique inthat is has no splice variants. Table 1 de-scribes the binding specificities of theFGF family that are known to induceangiogenesis to the various FGFRs.

All members of the FGF family arestructurally homogeneous, but the spec-ificity of their growth-promoting activityvaries. FGF1 and FGF2 differ from allother FGFs in that they lack a signalpeptide that would otherwise channeltheir secretion into the extracellular ma-trix by the classic endoplasmic reticulum/Golgi/vesicle pathway. FGF1 and FGF2have been studied and used the most inclinical trials to induce therapeutic an-giogenesis, but animal models and chickchorioallantoic membrane assays havedemonstrated that FGF3, FGF4, and

FGF5 also have a positive regulatory ef-fect on angiogenesis (Giordano et al.1996, Yoshida et al. 1994).

FGF4 and FGF5 share a 42% and50% amino acid sequence homology, re-spectively, with the core region of FGF2.The angiogenic capacity of the remain-ing FGFs has not been defined. One ofthe difficulties in ascribing a particularbiologic effect, especially a complex onesuch as angiogenesis, to a particularFGF is the high degree of compensatoryactivity among FGF family members. Itis likely that variations in receptor-bindingspecificity, differing tissue distribution,and temporal patterns of expression ofboth FGF and FGFR may well accountfor the subtle differences in their bio-logic activities.

For example, despite its diverse bio-logic profile, FGF2 gene disruption inmice merely leads to decreased vascularsmooth muscle cell contractility, lowerblood pressure, and thrombocytosis (Zhouet al. 1998). Although, as noted above,redundancy among the FGFs likely ac-counts for such a mildly altered pheno-type, compensation by FGF1 does notexplain the FGF2

�

/

�

phenotype (Milleret al. 2000). Likewise, disruption ofFGF1 or FGF5 genes also leads to onlymild changes in phenotypes. At thesame time, disruption of FGFR1 resultsin early embryonic lethality (Deng et al.1994, Yamaguchi et al. 1994).

We will briefly consider the afore-mentioned FGF2 (basic FGF) as a proto-typic FGF. It is a 16.5 kDa 146-amino-acid peptide that binds with high affinity(10

�

9

M) to cellular and extracellularmatrix heparan sulfates and with evenhigher affinity (10

�

11

M) to its specific ty-rosine kinase receptors (Nugent andEdelman 1992). Heparan sulfate bind-ing of FGF2 prolongs the effective tissuehalf-life of the peptide and enhancesthe binding to its high-affinity receptors(Rosenberg et al. 1997). FGF2 is presentin significant amounts in most normaltissues, including the myocardium(Casscells et al. 1990). It is upregulatedby hemodynamic stress and, to some ex-tent, by hypoxia (Bernotat-Danielowskiet al. 1993, Kuwabara et al. 1995). Themechanism of hypoxia-induced increasein FGF2 expression is not clear: it is notmediated by hypoxia inducible factor 1

�

(Fang et al. 2001), but appears to involveJnk-1 signaling (Le and Corry 1999). Re-cent in vitro studies have demonstrated

that hypoxia enhances endothelial cellsensitivity to FGF2 by upregulation ofheparan sulfate FGF2 binding sites (Liet al. 2002).

At the heart of its angiogenic activityis FGF2’s ability to stimulate prolifera-tion and migration of endothelial cells invivo (Carmeliet 2000, Ware and Simons1997). In addition, the growth factoralso has cardioprotective antiapoptoticactivity (Cuevas et al. 1999, Hampton etal. 2000, Yanagisawa-Miwa et al. 1992).Because it is mitogenic for smooth mus-cle cells and macrophages, FGF2 inducesgrowth of larger “collateral” vessels, pos-sessed of fully formed media and adven-titia, in addition to stimulating capillarygrowth. Such vessels may be more suit-able as functional blood-carrying “con-duits” to the ischemic territory, in contrastto mere neocapillary formation (Scholzet al. 2001). It also may be involved inthe induction of angioblast differentia-tion and migration (Poole et al. 2001).

Despite appreciable amounts of FGF2protein found in normal tissues, there isa lack of significant angiogenesis underphysiologic conditions. The low endoge-nous expression levels of FGF2 receptors,including FGFR1 and syndecan-4, par-tially account for this phenomenon. Inaddition, FGF2 may be sequestered inthe extracellular matrix by binding to theheparan sulfate-carrying proteoglycan,perlecan, and also to heparan sulfatechains shed from cell surfaces, and thusmay not be available to serve as an effec-tive ligand for its signaling receptors. Al-though levels of FGF2 are elevated withinthe plasma (Cuevas et al. 1997) and peri-cardial fluid (Fujita et al. 1996) of pa-tients with acute coronary syndromes,the significance of its contribution tosubsequent neoangiogenesis within theischemic territory is not clear.

The highly encouraging results thatemerged from the use of FGF1, FGF2,FGF4, and FGF5 in preclinical studies incanine and porcine models (for a review,see Post et al. 2001) led to several phaseI clinical trials in patients with severeischemic peripheral and coronary arte-rial disease (Table 2) that were predomi-nantly designed to determine safety andto establish the maximum tolerateddose by using a single- or double-dose-escalating regimen and a variety of de-livery strategies. Later studies examinedthe efficacy of FGF angiogenic therapy.These trials are discussed in turn below.

Table 1. Relative affinity of angiogenic FGFs for different FGF receptor isoforms

a

FGF1 FGF2 FGF4 FGF5

FGFR1: IIIb

1 0.6

0.16 0.04FGFR1: IIIc

1 1.04 1.02 0.59

FGFR2: IIIb

1

0.09 0.15 0.05FGFR2: IIIc

1 0.64 0.94

0.25

FGFR3: IIIb

1

0.01 0.01 0.01FGFR3: IIIc

1 1.07 0.69

0.12

FGFR4 1 1.13 1.08

0.07

Values in boldfaced type may represent an effica-cious interaction. FGF, fibroblast growth factor;FGFR, FGF receptor.

a

Adapted from Ornitz et al. 1996, p. 15,296. Used bypermission of the American Society for Biochemis-try & Molecular Biology.

118

TCM Vol. 13, No. 3, 2003

• Coronary Artery Disease (CAD) Clinical Trials

One of the earliest phase I studies(Schumacher et al. 1998) involved theintramyocardial injection of FGF1 at thesite of internal mammary artery–leftanterior descending artery anastamosisterritory during concomitant coronaryartery bypass graft (CABG) surgery. Angio-graphic techniques confirmed enhancedcollateralization and capillary prolifera-tion in the anterior wall of treated pa-tients compared with controls. However,in the absence of impaired perfusion,the functional impact of this therapycould not be assessed.

Another phase I clinical evaluation ofFGF2 therapy in the setting of CABG(Sellke et al. 1998) used slow-releaseheparin alginate microspheres (10 or100

�

g FGF) implanted within the epi-cardial fat overlying viable ischemicmyocardium deemed surgically unsuitedfor bypass. The 24 patients who met thecriteria were randomized at the time ofsurgery in a double-blinded fashion to10 low- or high-dose microspheres orplacebo. Nuclear and MRI perfusionscans were performed prior to hospitaldischarge and then again at 90 days(Laham

et al. 1999c). All patients in the100-

�

g FGF treatment group reportedno limiting ischemic cardiac symptomsat the 90-day evaluation, whereas three

of the seven control group patients ex-perienced persistent symptoms and twoneeded additional revascularizationprocedures. Nuclear perfusion imagingshowed a significant reduction in thesize of the ischemic target region in the100-

�

g FGF2 group, but not in the 10-

�

ggroup. Most importantly, the benefits ofFGF2 therapy were maintained after 3years of follow-up (Ruel et al. 2002).

The safety and feasibility of intracor-onary single-bolus FGF2 delivery wastested in two open-label dose-escalationstudies. One study (Unger et al. 2000) re-cruited 25 patients with subcritical cor-onary artery disease and randomizedthem to increasing doses (3 to 100

�

g/kg) of FGF2 or placebo. In the other trial(Laham et al. 1999b), 52 patients with se-vere coronary artery disease who weresuboptimal candidates for conventionaltherapeutic approaches received intrac-oronary infusions of FGF2 ranging from0.33 to 48

�

g/kg over 20 min. In both tri-als, FGF2 infusions were well toleratedwith systemic hypotension becomingdose limiting at 48

�

g/kg. Transient mildthrombocytopenia and proteinuria oc-curred in some subjects at 30

�

g/kg.Clinical monitoring over 6 months inthe second trial documented mortalityin four patients: two sudden deaths inpatients with ejection fractions of 22%and 30%, one death following cardiac

transplant for progressive heart failure,and one from non-Hodgkin’s lymphomadiagnosed 8 days following FGF2 infu-sion (Laham et al. 2000). No significantlaboratory toxicity, including proteinuria,was observed. Angina frequency and ex-ertional capacity scores were improvedin the entire FGF2 patient population at2 and 6 months compared with baseline.The FGF2 patients also demonstrated a2.4 min improvement in treadmill exer-cise time, whereas MRI perfusion imag-ing demonstrated a significant reduc-tion in the size of the ischemic regioncoupled with enhanced left ventricularwall motion in the same territory. Theresults from both of these trials sug-gested that intracoronary infusions ofFGF2 could be tolerated and might pro-duce clinically significant benefits.

This claim was tested in 337 patientsenrolled in a multicenter, double-blind,phase II trial (Simons et al. 2002) thatexamined three different concentrations(0.3, 3, and 30

�

g/kg) of single intracoro-nary infusions of FGF2 versus placebocontrols [the FGF Initiating RevaScular-ization Trial (FIRST)] in patients withadvanced coronary artery disease. Effi-cacy was evaluated at 90 and 180 days byexercise tolerance test (ETT), myocar-dial nuclear perfusion imaging, SeattleAngina Questionnaire (SAQ), and Short-Form 36 (SF-36) Questionnaire. Ninety-

Table 2. Summary of FGF clinical trials

FGF FormulationDose(

�

g/kg) Delivery Design Patients

(n) Follow-up Primary endpoint Reference

FGF1 Peptide 10 im [I] open-label 20 12 weeks DS angiography Schumacher et al. 1998

FGF2 Peptide 0–100 ic, single [I] open-label 25 29 d ETT, angiography Unger et al. 2000

FGF2 Peptide 0.3

→

48 ic, single [I] open-label 52 29 d, 57 d, 180 d

SAQ, ETT, MRI Laham et al. 2000

FGF2 Peptide 0, 10, 100 Hep-alg [II] DBR 24 90 d, 33 mo CCS, SPECT Laham et al. 1999cRuel et al. 2002

FGF2 Peptide 0.3

→

48 ic, iv [I] open-label 59 29 d, 57 d, 180 d

ETT Udelson et al. 2000

FGF2 Peptide 0, 10, 30 ia single, double

[I] DBR 19 4 weeks, 24 weeks

Plethysmography Lazarous et al. 2000

FGF1 plasmid DNA

500–16,000

�

g im [I] open-label 51 12 weeks Calf arteriography Comerota et al. 2002

FGF2 Peptide 0, 0.3, 3, 30 ic, single [II] DBR 337 90 d, 180 d SAQ, ETT, SPECT Simons et al. 2002

FGF4 Adenoviral 3.3

�

10

8

–10

9

ic [I/II] DBR 79 30 d, 90 d ETT Grines et al. 2002

FGF2 Peptide 0, 0.3, 3, 30 ia, single, double

[II] DBR 190 90 d ABI Lederman et al. 2002

ABI, ankle-brachial index; CCS, Canadian Cardiovascular Society; DBR, double-blind, randomized; DS, digital subtraction; ETT, exercise tolerance test; FGF, fibro-blast growth factor; FGFR, FGF receptor; ia, intra-arterial; ic, intracoronary; im, intramyocardial (muscular); SAQ, Seattle Angina Questionnaire; SPECT, singlephoton emission CT.

TCM Vol. 13, No. 3, 2003

119

day follow-up data demonstrated thatall groups (both FGF2 and placebo)showed a significant improvement in ex-ercise tolerance compared with base-line. FGF2 treatment, however, was nobetter than placebo. Exercise toleranceonly marginally improved by less thanor equal to 10 s at the 6-month assess-ment point, and the difference betweenFGF2 treatment and placebo remainedinsignificant because of sustained andcontinued improvement within the pla-cebo group (Simons et al. 2002).

At 3 months, FGF2 treatment was as-sociated with significant improvementin various quality of life parameters(SAQ, SF-36, angina class); however, by6 months these differences disappearedbecause of ongoing improvement in theplacebo group. Nuclear imaging also re-vealed no significant improvement inthe size of the ischemic territory, al-though patients with ischemia in therest nuclear imaging study (hibernatingmyocardium) demonstrated a signifi-cant reduction in the size of this defect.Interestingly, subgroup analysis of thestudy revealed that the benefit of FGF2treatment was most prominent in highlysymptomatic patients (baseline anginafrequency score

40 or Canadian Car-diovascular Society [CCS] score of III orIV). Although the results fell short ofexpectations, several important lessonscan be extracted from this importanttrial. Categorizing patients who are likelyto respond to exogenous growth factortherapy will be crucial in selecting can-didates for future trials. The extent andprevalence of the placebo effect in thispatient population also surpassed expec-tations and set the precedent that evalua-tion of efficacy is possible only in double-blind trial format. However, the absenceof sudden death or excess mortality andlack of potential toxicity secondary topathological angiogenesis reiterated therelative safety of FGF2 therapy (Simonset al. 2002).

Intracoronary administration of anadenoviral (Ad)-mediated FGF5 gene tosuccessfully alleviate myocardial ischemiaand improve function in a porcine ameroidconstrictor model (Giordano et al. 1996)provided the rationale for the AngiogenicGene Therapy (AGENT) trial (Grines et al.2002). The AGENT trial was a phase I/IIdose-escalating (3.3 � 109 to 3.3 � 1010

total viral particles) study that enrolled79 patients with chronically stable is-

chemic cardiac symptoms who wererandomized to a single intracoronary in-fusion of AdFGF4 (n 60) or placebo(n 19). These patients had moderatelylimiting disease (CCS II or III), with atleast one major coronary artery lessthan 70% narrowed and thus amenableto further interventional revasculariza-tion, rather than the “no-option” pa-tients that are typical of other coronaryangiogenesis trials. ETT constituted theprimary endpoint for efficacy and wasassessed at 4 and 12 weeks.

Overall analysis of all AdFGF4-treatedpatients showed a nonsignificant trendtoward improved exercise tolerance at 4weeks. Subgroup analysis demonstratedseveral interesting findings. ETT im-provement was significant at 4 weeks forpatients treated with 109 total viral parti-cles. Statistical significance also wasachieved when only patients with a base-line ETT of less than or equal to 10 minwere evaluated, at both 4 and 12 weeks.Patients with a low initial neutralizingantibody titer to adenovirus (�1:100)also fared significantly better, comparedwith those who had a higher titer. How-ever, a follow up AGENT2 trial did notdemonstrate this relationship (M. Wat-kins personal communication).

Most of the patients who received ac-tive therapy demonstrated a rise in anti-adenoviral antibodies. This fact has im-plications for repeated adenoviral genedosages, administered over time, thatmay lose their efficacy (Gilgenkrantz etal. 1995). Side effects consistent withthe propensity of adenovirus to induceflu-like syndromes and transient dose-related hepatic toxicity rarely were ob-served and thus confirmed the safety ofthis approach. Nevertheless, vigilancefor potential toxicity should be main-tained in the phase II/III trial. Two ma-lignancies were diagnosed in patients re-ceiving AdFGF4 infusions (a metastaticcolon carcinoma and brain tumor), butboth were considered to be unrelated toAdFGF4 treatment, in part, because oftheir negativity for the adenovirus.

• Peripheral Arterial DiseaseClinical Trials

Intra-arterial recombinant FGF2 wasadministered to patients with symptomsof claudication and an ankle-brachialindex (ABI) of less than 0.8 in a double-blind, dose-escalation phase I trial (Laz-

arous et al. 2000). Patients were ran-domly assigned to placebo (n 6), 10�g/kg (n 4), 30 �g/kg once (n 5), or30 �g/kg FGF2 on 2 consecutive days(n 4). Strain gauge plethysmographydemonstrated a significant increase incalf blood flow at 1 and 6 months in pa-tients treated with the higher FGF2 dose,with no apparent change in the placebogroup. FGF2 was well tolerated withoutprovoking any adverse morbidity.

Another phase I trial (Comerota et al.2002) reported the safety of using intra-muscular FGF1 gene injection (nakedplasmid DNA) in 51 patients with ad-vanced peripheral arterial disease (ABI �0.4) who were experiencing ischemicrest pain that was not salvageable byconventional surgical measures. Dosesfrom 500 to 8000 �g of FGF1 plasmidwere administered as a single or repeatedinjection. No adverse event deemed re-lated to the treatment was reported.Postprocedural serum FGF1 protein lev-els were elevated in only one patient,suggesting either adequate retention bythe targeted muscle or insufficient up-take and translation of the FGF1 gene.Preliminary clinical outcome data from15 patients showed a significant in-crease in the ABI being documented at 2and 3 months that, unfortunately, wasnot sustained at 6 months. Aggregate ul-cer size showed a marked and signifi-cant reduction in size in nine patients.

The Therapeutic Angiogenesis withRecombinant Fibroblast Growth Factor-2for Intermittent Claudication (TRAFFIC)study randomly assigned 187 patientswith infrainguinal atherosclerosis andclaudication to either single- or double-dose bilateral intra-arterial infusions ofrecombinant FGF2 or placebo (Leder-man et al. 2002). Treadmill performance,the primary endpoint of the study, yieldeda 0.6-min increase in the placebo-treated group, a 1.77-min increase in thesingle-dose-treated group, and 1.54-minincrease in the double-dose treated groups,but these differences did not achieve sta-tistical significance at 90 days (P .075,analysis of variance). Improvement inpeak walking time was maintained at180 days, but remained statistically in-significant because of continued im-provement in the placebo group. ABI forpatients in both the single- and double-dose groups improved only slightly at 90days compared with baseline. Perceivedquality of life, as determined by the SF-

120 TCM Vol. 13, No. 3, 2003

36 Questionnaire, was not enhancedwith FGF2 treatment. No significanttoxicity was seen, aside from transientproteinuria, which was dose related andpreferentially affected the diabetic pop-ulation. This trial was an important stepforward in angiogenesis therapy for pe-ripheral artery disease (Donnelly andYeung 2002).

• Placebo Effect

A striking consistency in all of the re-cently published double-blinded studiesis the profound and confounding pla-cebo effect, which diminishes the valueof the FGF treatment for the relativelysmall numbers of patients involved. Asimilarly pronounced placebo effect wasseen in the VEGF in vascular angiogene-sis trial (Henry et al. 1999)—which useda combined intracoronary/intravenousstrategy to deliver recombinant VEGF165

to patients with advanced ischemicheart disease, and in a percutaneous la-ser myocardial revascularization trial forpatients with refractory angina in the di-rect myocardial revascularization in re-generation of endomyocardial channelstrial (Leon et al. 2002).

The placebo effect is not limited to“soft” symptomatic endpoints but alsohas been observed in such “hard” end-points as positron emission tomography-,MRI-, and single photon emission CT-determined improvement in myocardialperfusion and function in these pa-tients. Thus, it is clear that this is a realbiologic phenomenon. Part of the ex-planation may lie in improved compli-ance of trial patients with their medicalregimens. The other contributing fac-tors may include increased physical ac-tivity and exercise, which may them-selves promote angiogenesis (Richardsonet al. 1999). However, the nature of thiseffect requires further study. Figure 1shows the “placebo creep” phenomenonexhibited by the placebo group, whichnullifies any significance that manifestsitself at the earlier time points in theFIRST and TRAFFIC trials.

• Pharmacokinetics of FGF and Growth Factor Delivery

The results of the clinical trials summa-rized above should be considered interms of the current understanding ofthe biology of angiogenesis and known

pharmacokinetic profiles of various de-livery modalities. Emerging experimen-tal data suggest that a sustained pres-ence of the growth factor in tissue isrequired development and maintenanceof newly formed vasculature (Dor et al.2002). The achievement of such a longpresence may require either sustained-release polymer in the case of proteintherapy or long-lasting gene therapy vec-tors. The current experience with plas-mids and adenoviral-based vectors sug-gests that the duration of expression israther short and may not be adequatefor long-term vessel maintenance (Yla-Herttuala and Martin 2000).

Biodistribution analysis of 125I-FGF2in a canine myocardial ischemia modelrevealed that only 3% to 5% of the origi-nal dose is retained within the myocar-dium 150 min after an intracoronary in-fusion, whereas the fraction was evensmaller and therapeutically ineffectualafter an intravenous infusion (Lazarouset al. 1997, Unger et al. 1994). This lowmyocardial retention rate is probablydue to “first-pass” pulmonary metabo-lism, by virtue of FGF-binding heparansulfate receptors, which significantly re-duce the myocardial FGF exposure. La-ham et al. (1999b) noted that by 24 h, themyocardium was relieved of essentiallythe entire dose, even after intracoronaryinfusion. Myocardial uptake was similarwith Swan Ganz and intravenous ad-ministration, suggesting that limitingthe delivery of the total drug dose to apulmonary segment, rather than the en-tire pulmonary bed, does not saturatethe available pulmonary binding sites(Lazarous et al. 1997).

More recently, the pharmacokineticsand pharmacodynamics of a single dose(0.33–48 �g/kg) of intracoronary or in-travenous infusion of recombinant FGF2were examined in 66 patients with se-

vere coronary artery disease (Bush et al.2001). Plasma concentrations declinedrapidly over the first 4 h, which was fol-lowed by a more prolonged terminalelimination phase for all doses tested.The mean elimination half-life was mea-sured to be 7.6 h, in contrast to an ear-lier study that reported it to be 1.4 h(Unger et al. 2000). Furthermore, theconcentration-time profiles were com-parable for intracoronary or intravenousinfusion of 36.0 �g/kg, indicating simi-lar systemic exposure for the two routes.FGF2 peak plasma concentration in-creased proportionally with dose, indi-cating linear pharmacokinetics. Greatersystemic exposure to FGF2 was observedwhen heparin was administered in closertemporal proximity to the FGF2 infu-sion, consistent with slower clearance ofFGF2–heparin complexes.

These observations suggest that a sin-gle intracoronary administration (or intra-arterial, in the case of peripheral arterydisease trials)—although effective inyoung, healthy, growing animals—is un-likely to produce a sustained benefit inpatients. The results of the FIRST andTRAFFIC trials are fully consistent withthis notion.

The efficacy of intracoronary injec-tions for adenoviral delivery is more con-troversial, with some studies (e.g., Grineset al. 2002) reporting very high first-passextraction, and others (e.g., Lazarous etal. 1997) not confirming this observation.

Intramyocardial or, in the case of pe-ripheral artery disease trials, intramus-cular administration, remains the chiefalternative that has yet to be tested ex-tensively in clinical trials. The advan-tages of this delivery modality includehigh initial retention of protein, plas-mid, or an adenovirus, and relativelyslow washout (Kornowski et al. 2000).This delivery technique, especially in the

Figure 1. The incremental “creep” effect of placebo at 90 and 180 days in the FGF InitiatingRevaScularization Trial (FIRST) and the Therapeutic Angiogenesis with Recombinant Fibro-blast Growth Factor-2 for Intermittent Claudication (TRAFFIC) trials.

TCM Vol. 13, No. 3, 2003 121

heart, requires advanced technical capa-bilities [injection catheters, left ventric-ular (LV) mapping catheters] and signif-icant operator experience. Nevertheless,this is a promising approach that willundergo extensive preclinical and clini-cal evaluation.

Other delivery modalities are less ap-pealing. Although intrapericardial deliveryhas a number of theoretic advantages—including direct contact with epicardialcoronary vasculature and slow washout—and is technically feasible even in pa-tients without pericardial effusion, it isunlikely to become a significant deliverystrategy because of high prevalence ofprior cardiac surgery in patients referredfor therapeutic angiogenesis trials (Si-mons et al. 2000).

The sustained-release polymer-baseddelivery, although meeting many of thebiologic challenges for an effective treat-ment modality and an apparent clinicalsuccess in clinical trials (Laham et al.1999a, Ruel et al. 2002), presents a prac-tical difficulty because of the need foropen-chest delivery. A successful devel-opment of catheter-based injectablepolymers may overcome this problem.

• Long-Term Safety

There is a relative paucity of long-termfollow-up safety data from the numer-ous phase I/II angiogenic gene therapytrials. Preclinical investigations have un-covered the potential for deleteriousside effects with exogenous angiogenictherapy such as hemangioma formation(Lee et al. 2000); neovascularizationwithin atherosclerotic plaques, whichpromotes their instability (Celletti etal. 2001); latent undetectable tumors;neointimal formation (Nabel et al. 1993a);and accelerating retinal vasculopathy.Furthermore, human coronary atherec-tomy specimens display strong correla-tions among smooth muscle cell prolif-eration, lesion “instability,” and FGF1and FGF2 expression (Flugelman et al.1993). These concerns are addressed bycareful patient selection before trial en-rollment and, consequently, toxicity fearshave not manifested to any significantdegree to date in any of the trials. How-ever, this does not obviate the need forlonger-term follow-up information onsuch patients. Indeed, such informa-tion is essential to better understandthe nature of the profound placebo ef-

fect that has been observed in the im-mediate follow-up period of severalclinical trials.

• Conclusion

Trials investigating FGF-mediated thera-peutic angiogenesis are subject to someof the criticisms that have plagued othercandidate angiogenic growth factors.Monogene therapy, which has proved tobe reasonably efficacious within animalmodels, has not had the same impact inhumans. FGFs stimulate only a singleangiogenic cascade and thus are not thebiologic ideal to replicate a complexprocess that involves an orchestratedinterplay of numerous growth factors,cytokines, and matrix-binding molecules.The disappointing results of the recentFGF2 clinical trials, in conjunction withthe conflicting basic science data onlyserve to emphasize the complexity of theangiogenic process. There is reason,however, for cautious optimism. Evolvingmolecular technologies, such as gene-expression profiling of the “at-risk” popu-lation, may be able to stratify patients ac-cording to the deficiencies within thespectra of growth factors, cytokines, andsignaling molecules that contribute toangiogenesis. Patients whose native col-lateral response to chronic cardiac is-chemia is inadequate comprise a hetero-geneous pool and such technologiesmay lead to a clearer identification ofwhich patients would benefit from mo-lecular angiogenic strategies. The use ofendothelial precursor cells as targetedvectors for gene delivery also holdspromise to boost this evolving field oftherapeutic angiogenesis.

• Acknowledgment

Supported in part by NIH grants HL63609 and HL 53793.

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