Therapeutic angiogenesis in cardiovascular disease

REVIEWS

The development of drugs capable of stimulatingrevascularization of underperfused tissues remains anexciting but unrealized goal in cardiovascular thera-peutics. Although the past decade witnessed vigorousgrowth of research and development in this area, noeffective drugs that promote revascularization havebeen approved for use, because the trials completed todate have failed to show unequivocal efficacy of thetested agents.

Recently, much has been learned about the processof new vessel growth and enlargement, the character-istics of agents that might generate new blood vesselsin patients, and the nature of clinical investigationsthat could show efficacy and safety of this novel classof medications. This review will focus on the newknowledge derived from the completed clinical trials,and the changes that could be introduced into the dis-covery and development process to improve thechances of identifying new drugs that promote revas-cularization. We will first summarize the current stateof clinical experience and then discuss three principalissues that need to be resolved: the identification ofagents that promote growth and remodelling of largervessels (arteriogenesis) rather than smaller vessels(true angiogenesis); the establishment of the requiredlength of drug exposure in vivo and optimal means ofdrug delivery; and the selection of patients, clinicaltrial end-points and indications for those therapies.

Completed therapeutic angiogenesis trialsMore than a decade of preclinical studies have pavedthe way for clinical testing of the concept that newvessels could be created to reduce ischaemia. Despiteour ability to use the angiogenic growth factors torestore blood flow and function to ischaemic tissues inmice, dogs and pigs (FIG. 1), the translation of thisexperience into clinical practice has been disappointingfor a few recurring reasons.

Although the initial series of open-label, uncon-trolled studies reported spectacular successes, subse-quent double-blind, randomized, controlled trials(TABLE 1) were far more sobering. The first trial thattested agents in the form of recombinant proteinsemployed a combination of intracoronary and intra-venous vascular endothelial growth factor (VEGF)

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protein infusions (VEGF in Ischaemia for VascularAngiogenesis (VIVA) trial), which produced no signifi-cant benefit in treated patients in terms of exercisetolerance, myocardial perfusion, angina symptoms orfunctional class at 60 days. A trend in favour of high-dose VEGF

165was seen at 120 days1, however. The FGF

Initiating Revascularization Trial (FIRST) tested a singleintracoronary fibroblast growth factor-2 (FGF2) infu-sion; significant improvements in functional status andsymptom class were observed with FGF2, but there wasno benefit compared with placebo in terms of exercisecapacity and myocardial perfusion 90 days later2.A small

THERAPEUTIC ANGIOGENESIS IN CARDIOVASCULAR DISEASEMichael Simons* and J. Anthony Ware‡

Despite considerable progress in the management of ischaemic cardiovascular disease during thepast three decades, there remains a significant population of patients who are not served well bycurrent treatment approaches. Stimulating revascularization in ischaemic regions is an attractivenovel therapeutic strategy, and several angiogenic agents anticipated to have the potential toachieve this goal have been clinically evaluated in recent years. However, as yet none have shownsufficient efficacy to be approved. Here, we consider the key findings from the completed clinicaltrials of therapeutic angiogenesis in cardiovascular disease, and discuss possible changes to theway in which such agents are developed that could improve the chances of success.

NATURE REVIEWS | DRUG DISCOVERY VOLUME 2 | NOVEMBER 2003 | 1

*Angiogenesis ResearchCenter and Section ofCardiology, Departments ofMedicine and Pharmacologyand Toxicology, DartmouthMedical School, Lebanon,New Hampshire 03756,USA.‡Cardiovascular ResearchEli Lilly & Co, Indianapolis,Indiana 46285, USA.Correspondence to: M.S.e-mail: [email protected]:10.1038/nrd1226

CLAUDICATION

A condition in which crampingpain in the leg is induced byexercise, typically as a result ofobstruction of the arteries.

ANGIOPLASTY

Catheter-based repair orunblocking of a blood vessel,such as a coronary artery.

RESTENOSIS

A re-narrowing or blockage ofan artery at the same site wheretreatment, such as an angioplasty,has already been performed.

2 | NOVEMBER 2003 | VOLUME 2 www.nature.com/reviews/drugdisc

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have failed to provide clear-cut evidence of therapeuticeffectiveness. Although there are tantalizing hints ofefficacy, on the whole the results fall far short of thoserequired for regulatory approval and clinical accep-tance of a new therapeutic agent. Nevertheless, webelieve that the limited successes noted above stronglysupport the concept that therapeutic angiogenesisagents can be of benefit to patients with cardiovasculardisease. We suggest that changes in our approach todiscovery and development could increase the likeli-hood of identifying a successful agent to promoterevascularization.

At first glance, the strategic goal for a research pro-gramme to improve on these results would seem to bestraightforward. Agents that induce revascularization inpreclinical models have been identified and brought toclinical testing, and have been found to have modestefficacy and a very reassuring safety profile; so, onereasonable approach is to optimize these agents to showimproved performance in preclinical testing, and thenreturn to the bedside for clinical testing. On furtherconsideration, however, it becomes apparent that theproblem is more complex than this. At least three con-siderations come to mind. First, preclinical models relyingon young, healthy animals might have biology that isfundamentally different from that of older humanpatients with advanced atherosclerosis. Second, theagents tested so far might be more appropriate forcapillary growth (true angiogenesis), rather than growthor enlargement of a larger conduit (arteriogenesis (FIG. 2));see below), and therefore be incapable of promotingdramatic increases in blood flow. Last, it is possible thatthe available agents are capable of eliciting the responsesseen in preclinical models in patients, and that improve-ment in time of exposure to the targeted tissue wouldproduce beneficial results.

Such changes in the level and/or time of exposuremight require different forms of delivery than therecombinant protein, given as a single bolus, used formost of the studies so far. Either polymer-based deliveryor a gene-transfer mechanism would be likely to alterthe pharmacokinetics of the agent. Alternatively, a sepa-rate but related solution to produce favourable pharma-cokinetics might be local drug delivery into the targetorgan by direct injection. Another option that could beconsidered is a small molecular (chemical) entity thatmight be given systemically for a short period of time;administration could be repeated in cycles as necessaryfor ischaemia. It soon becomes apparent that, for anygiven revascularization agent, there are several potentialcombinations that could be assessed in preclinical modelsand clinical testing. We believe that three changes in ourfocus are essential to the successful development of anagent that promotes revascularization, and we brieflysummarize each of these suggestions below.

Focus on arteriogenesisWhereas the development of vasculature in the course ofembryonic growth is beginning to be well understood,comparatively little information is available about theprocesses responsible for vessel growth and maintenance

randomized trial of polymer-released FGF2 implantedat the time of coronary artery bypass surgery in theunrevascularized myocardium demonstrated a signifi-cant improvement in perfusion and symptom statusafter 90 days in the high-dose FGF2 group comparedwith controls3. Interestingly, the benefit of FGF2 therapywas still seen at a three-year follow-up4. Finally, in thefirst randomized trial that examined results in patientswith peripheral vascular disease, a single but not a double(30 days apart) intra-arterial FGF2 infusion in patientswith CLAUDICATION improved peak walking time at 90days compared with placebo controls. No differencesamong the treatment groups were detected after sixmonths, however5.

Controlled trials involving gene transfer also have hadpromising, but not overwhelmingly positive, clinicalresults. Intracoronary infusion of an FGF4-encodingadenovirus (Ad) demonstrated a trend towards improvedexercise performance that was especially significant inpatients with low titres of anti-adenoviral antibodies6.A small trial of intramyocardial injection of VEGF2plasmid demonstrated a significant improvement inangina symptom class and trends towards improvementin exercise performance and myocardial perfusion7.Neither plasmid nor adenoviral-based VEGF

165gene

transfer into the wall of the coronary artery at the time ofANGIOPLASTY had any effect on RESTENOSIS, but patientsreceiving Ad-VEGF

165injection demonstrated smaller

perfusion defects on nuclear scans8.So, the initial series of double-blind randomized

trials (TABLE 1) utilizing the two most extensively studiedfamilies of angiogenic growth factors, VEGF and FGF,

a b

Figure 1 | Therapeutic angiogenesis. a | Therapeutic angiogenesis in two different fields (topand bottom). Histological appearance of newly formed vessels in the ischaemic porcine.Myocardial tissues were injected with VEGF165 protein. Three weeks later the animals werekilled and tissue sections were stained with anti-vWF antibody. Note the presence of largevascular structures lined with endothelial cells and devoid of media or adventitia layers. b | Angiogram of porcine coronary arteries. The left circumflex coronary artery was instrumentedwith an ameroid occluder (arrowhead) and FGF2-containing heparin-alginate pellets wereinserted around the occluder. Coronary angiogram performed three weeks later demonstratesthe presence of collateral vessels (arrows) fully reconstituting blood flow in the distal arterialsegment. Panel a adapted with permission from REF. 59 © The American Physiological Society(1996). Panel b adapted with permission from REF. 40 © Elsevier Science Ltd (2001). FGF,fibroblast growth factor; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.


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induce the growth of larger vessels (arterioles andarteries, a process termed arteriogenesis) is not estab-lished, but it seems to be less active in this regard thanare FGFs and PDGFs. One potential mechanism forarteriogenesis in these conditions is VEGF-mediatedrecruitment of circulating monocytes that then stimu-late a local arteriogenic process (see below). A secondpotentially important role for VEGF is in the mainte-nance of newly formed vasculature. Genetically alteredmice have revealed that newly formed vasculature com-pletely disappears after a limited VEGF exposure, butthat a longer duration of VEGF stimulation (10–14days) results in the production of mature vessels that donot resorb after withdrawal of the growth factor17.

Preclinical studies are largely consistent with theobserved limited efficacy of VEGF in stimulating signif-icant revascularization administered as a single agent.Although a transient benefit is observed in a number ofmodels, the absolute improvement is fairly small andthe longevity of the newly formed vasculature isunclear. Furthermore, other pharmacological propertiesof VEGF, including its propensity to induce severehypotension18 and tissue oedema, limit the dose thatcan be given systemically to below that likely to showefficacy19. These considerations are likely to limit thetherapeutic applications of VEGF to local delivery, andalso indicate that this growth factor is probably bestused in combination with agents that induce formationof larger vessels.

HIF-1α seems to be a more potent agent than VEGFby itself20, an indication that other genes activated by thetranscription factor favourably influence its therapeuticproperties. Which HIF-1α-induced genes mediate thisadditional response is not clear.

In contrast to ischaemia-driven angiogenesis, thefactors regulating arteriogenesis (FIG. 2) are very poorlyunderstood. As noted above, arteriogenesis refers to theprocess of growth of larger-size arteries consisting ofthree vessel wall layers. This can occur either via remod-elling of pre-existing small collaterals16 or via the growthof new vessels21. The relative contribution of these twoevents in either animal models or patients is unclear. Inarteriogenesis, the growth of new vessels occurs around

in mature adult tissues. Three principal responses, whichcan be categorized as true angiogenesis, arteriogenesis(FIG. 2) and vasculogenesis, seem to be involved in vesselgrowth, although their relative importance and under-lying molecular triggers and pathways remain unclear.

True angiogenesis, used here to describe capillaryproliferation in ischaemic beds, is the most understoodof the three. Tissue hypoxia is the major driver of thisprocess. Increased expression of hypoxia-induciblefactor-1α (HIF-1α) protein, a nuclear transcription fac-tor, is the primary molecular event stimulating angio-genesis. This increase in HIF-1α expression resultsprimarily from stabilization of the inherently unstablemature protein9–11. The cell type in which increases inHIF-1α expression occurs differs among various settingsbut predominantly includes pre-vascular cells, such aspericytes and cardiac or skeletal myocytes, because theendothelium itself rarely becomes ischaemic.

The increased expression of HIF-1α leads toincreased transcription of a number of genes involvedin angiogenesis, including VEGF, the VEGF receptorFLT-1 and angiopoietin-2, among others9. Thesechanges in gene expression predominantly activateVEGF-dependent endothelial cell proliferation.Signalling by other angiogenic growth factors, mostnotably FGFs12 and platelet-derived growth factors(PDGFs)13, which might stimulate smooth muscle cellsnecessary for support and stabilization of larger vessels,is not significantly affected. One exception to this is theincreased responsiveness of hypoxic endothelial cellsto FGF2 due to a HIF-1α-dependent increase inFGF2-binding heparan sulfate sequences14.

So, the primary physiological response to tissueischaemia is the local growth of capillaries. Althoughsuch a response is certainly useful in wound healing andother tissue repair processes, it is not clear whether it issufficient to compensate for ischaemia resulting from aproximal occlusion of a major arterial trunk, as is thecase with many patients with coronary or peripheralvascular disease. Indeed, theoretical considerationssuggest that a vast increase in the capillary bed size isrequired to compensate for occlusion of a singlemedium-size artery15,16. Whether VEGF itself is able to

Table 1 | Double-blind trials of therapeutic angiogenesis agents

Trial Trial type N Agent Administration Result References

VIVA Phase II 178 VEGF165 Protein infusion (IC, IV) ND at day 60; late 1improvement in VEGF group

FIRST Phase II 337 FGF2 Protein infusion (IC) Mixed results day 90; 2ND day 180

Hep-Alg Phase I/II 24 FGF2 Local in polymer Improvements at 90 day 3,4and three years

TRAFFIC Phase II 190 FGF2 Protein infusion (IA) Improvement at day 90; 5 ND at day 180

AGENT Phase I/II 79 FGF4 Adenovirus infusion (IC) Trend towards benefit 6

VEGF2 Phase I/I 19 VEGF2 Plasmid injection (IM) Symptomatic improvement 7

KAT Phase II 103 VEGF165 Protein, plasmid or Improved perfusion in 8adenovirus injection (IC) adenovirus group

IA, intra-arterial; FGF, fibroblast growth factor; IC, intracoronary; IM, intramyocardial; ND, no difference; VEGF, vascular endothelial growth factor.


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with angiogenesis or arteriogenesis.At present, the signif-icance of the contribution of these cells to ongoing neo-vascularization following ischaemia is unclear. Severalfactors contribute to the difficulty of assessing the contri-bution of endothelial precursor cells to arteriogenesis.One is the lack of agreement as to the definition of anendothelial precursor cell; numerous antigen markershave been proposed, but few data correlate the mark-ers with outcome27.Another is the effect of cell fusion, inwhich the nuclear contents of two cells fuse to create acell with a double chromosome content, which can con-fuse the interpretation of experimental results28,29. Thecirculating bone marrow cells seem to be particularlysusceptible to this fate; if such cells are labelled with afluorescent marker, then the appearance of the marker incells exhibiting characteristics of a differentiated cell(such as an endothelial cell) does not necessarily indicatethat trans-differentiation has taken place.

Despite the lack of understanding regarding thesecomplicated issues of cell origin and fate, much atten-tion has been focused on demonstrating the functionalbenefits of cell therapy. Unfortunately, these studies arealso difficult to interpret. For example, numerous inves-tigators have demonstrated that the infusion of bonemarrow cells (unselected or isolated populations)improves recovery in ischaemic models in lethally irra-diated mice, and further that the transplanted cells arefound in the forming vessels27. It is not clear, however, thata similar benefit would be observed in non-irradiatedanimals, when one considers the contribution ofmonocytes and monocyte-derived macrophages toarteriogenesis.

Another possible approach is to transfer autologouscells, which has been studied in larger animals30,31 and inclinical trials32–34. When analysing these studies, it isimportant to note that most of the emphasis has beenon the demonstration of organ-specific recovery, ratherthan the evaluation of new vessel formation. For example,improved recovery of myocardial function followinginjections of bone marrow cells into the infarct areamight signify the creation of new myocytes from bonemarrow precursors, the stimulation of vessel growth tothe hibernating myocardium with subsequent recruit-ment, or simply stiffening of the dyskinetic area withresultant improvement in overall systolic performance.

With these caveats in mind, it is nonetheless attrac-tive to consider the implications of the role of endothe-lial precursor cells in neovascularization in adult tissuefor pharmacological drug development. Potential targetsmight include agents that expand (in vivo or ex vivo)specific cell populations, and those that improve cellhoming to ischaemic areas. Alternatively, such cellsmight be used as biological foundries to deliver growthfactors to a targeted tissue. The practical feasibility ofturning such strategies into viable products remainsunclear, however.

The development of, and gaining of approval for, anarteriogenic agent will probably be affected by some ofthe conceptual changes in vascular biology noted above.For example, it might prove that therapies combiningtwo (or more) agents that act on different aspects of the

the site of occlusion of the proximal arterial feedingtrunk (epicardial coronary artery in the heart, large con-ductance artery in the leg). Careful studies have demon-strated the absence of tissue ischaemia in the locale ofthe arterial growth, thereby essentially excludinghypoxia and HIF-1α as the primary mediators.Alternative candidates might include the numerousblood-derived activated macrophages that are foundalong the course of the developing vasculature, indicat-ing that these cells might be intimately involved in theprocess. The role of monocyte-derived macrophages isfurther emphasized by the ability of monocyte chemo-attractant protein-1 (MCP-1) to induce arteriogenesis22–25

and by observations of depressed arteriogenic responsefollowing monocyte depletion26.

Although these observations establish the role ofmonocytes in arteriogenesis, the signal responsible forthe initiation of monocyte accumulation is not certain.Altered shear stress in vessels proximal to an occlusionhas been proposed as a one potential mechanism;experimental data for this attractive concept are not yetdefinitive, however.

Vasculogenesis, a term that originally referred to theformation of primary vasculature from precursor cellsduring embryonic development, has been used todescribe a similar process in the adult. The major distinc-tion, however, is that in adult tissues, circulating precursorcells can contribute to new vessel formation in concert

Blockage

a Remodelling

b New vessel formation

Figure 2 | Arteriogenesis. Collateral vessels (arteriogenesis) can develop around the site ofcoronary occlusion. Although the exact mechanism is not clear, there are two distinct possibilities.One option (a) for arteriogenesis is to proceed via remodelling of pre-existing vessels thatgradually enlarge to the point at which they can carry the bulk of blood flow. The second option(b) involves budding of new vessels from post-capillary venules on the adventitial surface of theoccluded artery that gradually expand and connect to the distal arterial segment. The excessvessels undergo apoptosis once sufficient flow has been established.


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scale up for broader testing. As discussed earlier, thereare concerns with regard to predictability, in that manyagents that have achieved spectacular results in thesemodels were much less impressive when used in humanpatients. Indications of the successful induction of newvessel formation in a preclinical model are morestraightforward than in the patient, because of the avail-ability of histological samples and the ability to performrepeated invasive measures, but the problem of selectinga surrogate for a beneficial histological effect persistsnonetheless. Many of the well-described assays weredeveloped to test angiogenesis inhibitors; althoughthey can be adapted for pro-angiogenic substances, theymight not detect molecules that would be expected topromote arteriogenesis.

Optimal time course of growth factor therapyOne of the most important lessons from the currentgeneration of clinical trials, and ongoing basic research,has been the recognition of the need for prolonged tissueexposure to the growth factor for the development ofrobust and sustained neovascularization39. Although anumber of animal studies demonstrated the ability ofa single bolus of growth factor to induce vigorous newvessel formation40, such an approach has not been highlysuccessful in clinical practice. One possible reason for thisdiscrepancy is that the prolonged presence of a growthfactor might be required to secure the survival of newlyformed vasculature, as noted above17. The potential clini-cal significance of this finding should be assessed in largeanimal models of therapeutic angiogenesis with follow-up time longer than the typical three weeks41. The failureof sustained clinical benefit observed in some trials is inagreement with this premise. It is likely that the optimaltime of exposure will differ according to the particulargrowth factor, formulation and delivery strategy, and somight need to be established for each combination.

Another source of fundamental differences betweenpreclinical and clinical studies is the mechanism ofdelivery42. Although a variety of means can be used todeliver sufficient amounts of growth factors to stimulatenew vessel formation in animal models, the options inthe clinical setting are much more limited. Only thesimplest of delivery options (that is, systemic and intra-coronary injections) have been extensively tested so farin patients. This state of affairs might, paradoxically,have stemmed from the ease of cure with single-bolusinjections in young, healthy pigs. The most convenientmethod of administration to patients is by intravenousinfusion, but for presently available angiogenic agentsthe use of this route is complicated by dose-limitinghypotension and concerns regarding inappropriateangiogenesis resulting from systemic exposure. Theavailability of an agent that could be administered intra-venously without these limitations would be a majoradvance for the field.

Other potentially more effective methods of delivery(FIG. 3), such as local intramyocardial or intramuscularinjections, pericardial delivery or sustained-releasepolymers, have only been tested in small trials or havenot been clinically tested at all. It is probable that those

revascularization mechanism are more effective in pro-moting improved blood flow than either alone. Such apossibility could complicate clinical testing and marketentry. Gaining regulatory approval for such a combi-nation, assuming that neither is itself approved, is anadditional consideration. It is likely that the combina-tion of drugs will have to be shown to be more effectiveor safer than either alone.

Rather than using combinations of already testedgrowth factors, a second avenue towards improvingblood flow is to discover new agents with the promiseof greater efficacy while retaining the good safety pro-file of the current agents. Of course, the considerationsof drug form, mode of delivery and the ideal pharmaco-kinetic properties (discussed in the next section) mustbe established for these agents as well. One possibility isto use certain proteins that can operate as ‘master geneswitches’ and stimulate the activity of multiple growthfactors, thereby achieving with a single agent the biolog-ical effects gained with a combination of agents. HIF-1α,as noted above, is one such master switch, in that it hasthe ability to activate both VEGF and angiopoietin-2pathways. Other examples include hepatocyte growthfactor, which has both direct angiogenic effects as well asthe ability to activate VEGF35, and the PR39 peptide thatactivates both VEGF and FGF pathways36.

Another possibility is to use factors that can promoteboth angiogenesis and arteriogenesis (and perhaps vas-culogenesis) by virtue of their ability to activate multiplebiological processes. An example of these pleiotropicfactors is placental growth factor, which seems to stimu-late arteriogenesis (perhaps via monocyte recruitment)as well as promoting angiogenesis37. Another exampleis PDGF-B, which can promote capillary growth inaddition to the development of larger vessels38.

One can imagine a more open-ended approach tothe discovery of novel factors based on genomics, inwhich gene expression in patients with vigorous collat-eral vessel formation following vascular occlusion iscompared with that of patients who do not have newvessel formation, and the factors encoded by theseup/downregulated genes are investigated and tested. Asimilar approach might be taken at the protein levelwith the possibility of obtaining tissue from skeletalmuscle biopsies, circulatory monocytes or pericardialfluid at the time of aorta coronary bypass grafting.

Validating a candidate gene or protein as an agentthat is likely to promote revascularization requires test-ing in several assays, both in vitro and in vivo. In vitroassays will often include the measurement of endothe-lial cell proliferation, migration and tube formationon an artificial matrix. There are many in vivo assaysthat have been utilized for further validation. Those thatwould seem to best represent the clinical pathophysiol-ogy of ischaemic vascular disease are the hindlimbischaemia models (usually rabbit, rat or mouse) andthe ameroid constrictor coronary ischaemia model(usually dog or pig). Several caveats should be appliedto these models. In the ameroid model in particular, thelength of time required to test an individual agent undera single circumstance is extensive, and it is difficult to


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use cells as a means of delivery following ex vivo genetransfer. Furthermore, injections of a large number ofcells, perhaps regardless of their origin (bone marrow,skeletal muscle, adipocytes and so on) might serve as ameans of delivering large quantities of multiplegrowth factors.

Patient selection and clinical trial end pointsThe choice of patient populations and clinical end-points for the testing of agents promoting vasculargrowth is crucial for the successful development of thisclass of drugs. The selected strategy should balance notonly biological and clinical factors, but also the require-ment for regulatory approval. As no drugs have yet beenapproved for such therapeutic revascularization, all con-clusions about what is likely to be required for suchapproval are conjectural at this point. One approachcould be to test an agent and show benefit in a patientpopulation for which there are at present only limitedtherapeutic options. Understandably, the regulatory agen-cies have been more receptive to drugs that offer the pos-sibility of improved clinical outcomes for such patients.Although safety remains important for such drugs, thepresumed benefit to patients for whom there is no ade-quate therapy outweighs the risk of adverse events thatmight halt the development of drugs for conditions inwhich this is not the case. One such group of patients isthose with severe myocardial ischaemia that cannot bealleviated with percutaneous coronary intervention orbypass grafting. Another is peripheral arterial occlusivedisease, for which medical treatment is less than satisfac-tory in many patients. Other more broad patient popu-lations might also be candidates for therapy. It is likely,for instance, that a therapy shown to be effective inpromoting arteriogenesis would be attractive forpatients who are also candidates for angioplasty andbypass surgery; however, before an agent is approved forthose purposes, the standard of benefit will probably bemore stringent, either in terms of required clinicaloutcomes or safety compared with the current therapies.

A major consideration in patient selection isprompted by the clear difference between the ease ofinduction of an angiogenic response in young, healthyanimals versus the challenge of inducting a similarprocess in elderly, no-option/poor-option patients withextensive atherosclerotic vascular disease. These individ-uals often have advanced disease and have failed stan-dard forms of therapy. This selection probably ‘chooses’individuals whose natural revascularization mecha-nisms have failed, and who therefore might be particu-larly resistant to growth-factor-based therapies. Thisconcept has not been established definitively, but anobservation that supports this point of view is that theonly randomized trial to select a broad range of patientswith coronary artery disease, rather than the no-optionpopulation, demonstrated the most promising results todate, even using an intracoronary delivery strategy6.

In a broader sense, all studies conducted so far havedemonstrated a high variability in response amongpatients, indicating that some undetermined biologicalfactors might influence the ability of patients to

organizations developing growth factors might need tocollaborate with catheter-based delivery or device com-panies, despite cultural differences and fears of a morecomplex clinical testing and regulatory approval path.

Careful preclinical studies have demonstrated thatsingle injections of bolus protein43 or adenovirus44–46

into coronary arteries result in the miniscule depositionof material and a very short residency time in tissues.Although a number of strategies can be used in animalmodels to increase the efficiency of intracoronary genetransfer, such as disruption of endothelial barrier45,47, thefeasibility and safety of such approaches in humansremains to be established.

Intramyocardial approaches (FIG. 3) offer consider-ably better transfer and retention rates for both drugsand proteins, although the problem of incompleteretention of material after injections persists48, presum-ably due to a back-leak along the needle channel,absorption into lymphatics and blood washout. Manyunknowns remain, including whether non-fluoroscopicguidance to a specific injection site is required, whichmyocardial zone (that is, normal, border or ischaemic)should be the target, and what technical characteristicsof the delivery system (for example, needle size, length,shape and so on) are ideal. As an illustrative example ofthese considerations, the myocardial zone selectedmight affect the dose of the revascularization agentrequired. The efficiency of gene transfer seems to bereduced in ischaemic tissues, and ageing myocytesdemonstrate decreased efficiency of adenovirus-mediatedgene expression49. Likewise, protein half-life could besignificantly reduced in ischaemic tissues, especiallyduring ongoing tissue resorption or repair processes.

One delivery strategy that warrants consideration ispericardial delivery, which takes advantage of slowwashout from this space and the deposition of the deliv-ered agent near epicardial coronary arteries. The peri-cardial delivery approach has been shown to be effectivefor both proteins40 and viruses50. This method has twomajor limitations: it is invasive, and it lacks applicabilityin patients without a functional pericardial space, whichincludes many who have undergone bypass surgery.Another method of potential interest is cardiac perfu-sion by retrograde delivery via the coronary sinus51. Thispromising delivery modality allows the selective infiltra-tion of targeted areas; furthermore, the pharmacokineticcharacteristics of both proteins and genes injected bythis means seem to be at least comparable to intramyo-cardial injections. The major obstacles to overcome forits widespread use are the anatomic variability of thehuman coronary sinus that increases the technical com-plexity of this approach and frequent vein-to-veinshunts that interfere with targeted drug delivery, alongwith its invasiveness.

As noted above, cell-based therapies have recentlyreached the state of clinical testing. These approachescan be divided into those using precursor cells of specifictissues in the hope that these can reverse the existingdamage (for example, endothelial precursors to stimu-late vessel growth, myocyte precursors to replenish lostmyocytes and restore cardiac function), and those that


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factors. To date, however, we lack biomarkers that arecapable of predicting such a response, which wouldallow for a more uniform patient selection. The searchfor such biomarkers and their validation as predictors ofoutcomes and therapeutic responses is one of the mosturgent issues facing the field.

In addition to patient selection, the successful devel-opment of these drugs, and their subsequent approval,requires a thoughtful selection of clinical end points54.A time-honoured end point for clinical testing is majoradverse cardiovascular events, a composite end pointthat includes death, myocardial infarctions and otherend points such as recurrent ischaemia and the neces-sity for intervention. The complexity of the patientpopulation means that achieving this end pointwould require large and expensive trials, even with anoptimistic view concerning the effect of promotingneovascularization.

As such, clinical end points have focused more on themeasurement of myocardial ischaemia, in terms ofthe time taken to develop myocardial ischaemia duringexercise testing, accompanied by non-invasive assess-ment of ischaemia. Tools with growing popularity andacceptance include questionnaires to assess quality oflife and the frequency of angina, such as SF-36 andSeattle Angina Questionnaire (SAQ)55. Whether posi-tive outcomes on these measures will be sufficient towin regulatory approval remains untested; as notedabove, the perceived benefit to patients will be bal-anced against the real or perceived risk to the patient,and the relative attractiveness of other, establishedmodes of therapy.

One of the biggest challenges in the successfuldevelopment of agents that promote arteriogenesis isthe identification of easily measured surrogate mark-ers of the effects of drugs. An ideal surrogate markerwould be a reliable indicator of the action of the drugon its purported target, as well being closely con-nected to the therapeutic goal of neovascularization.So far, there are few, if any, measures that allow inves-tigators to easily correlate an arteriogenic drug dose withthe desired effect, which has complicated dose selec-tion for many such agents. In terms of surrogatemarkers of efficacy, the clearest need is for a methodto image newly formed vessels repetitively, and/ormeasure perfusion in the target organ (such as theheart or leg). Angiography has long been the goldstandard for vessel detection and characterization, butits resolution does not always allow for the detectionand quantification of newly formed collaterals, and itsinvasive nature makes it impractical for repetitive use.A discussion of the technologies now under investiga-tion for non-invasive assessment of vascularity andperfusion, including magnetic resonance imaging(MRI), positron emission tomography (PET) andcontrast ultrasound is beyond the scope of thisreview. The establishment of one or more of thesemodalities as a generally available and reproduciblesurrogate for improved vascularity of the target organwould be a major step in facilitating the developmentof angiogenic drugs.

respond to angiogenic factors. A post hoc sub-groupanalysis of two FGF2 trials suggested that the sympto-matic severity of the disease largely determines themagnitude of response, in that patients with moresymptoms showed greater improvement with the thera-peutic agent. Furthermore, a study of patients withadvanced coronary disease, but different extents of col-lateral development, showed marked differences in theability of blood-derived monocytes to respond tohypoxia by upregulating HIF-1α levels52. Finally, angio-genesis inhibitors might influence resistance to therapy.Impairment of nitric oxide production, a commonoccurrence in advanced atherosclerosis, can stimulateangiostatin production with subsequent inhibition ofnative coronary angiogenesis53. If patients had signifi-cant variability in the levels of circulating inhibitors,then these unappreciated differences might furthercontribute to this issue.

As these considerations suggest, the biological differ-ences among patients might well be decisive in deter-mining the therapeutic response of patients to growth

a

b

g

f

e cd

d

Figure 3 | Drug delivery to the myocardium. Growth factors or genes can be injected in thehealthy myocardium proximal from the occlusion to stimulate collateral genesis either throughan intramyocardial or transvascular route (a). In the same way, watershed areas from whichprojected collaterals emerge can be targeted (d). To enhance angiogenesis, growth factors areinjected into the border of the infarct (b) or the centre of the ischaemic zone (c). After intra-myocardial injection, injectate can be lost by rapid washout through the venous and lymphaticsystem (e), back leakage through the injection tract (f) or because of inadvertent intra-ventricular injection (g). Adapted with permission from REF. 60 © W. B. Saunders (2003).


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symptomatic end points such as angina class, anginafrequency or exercise capacity, but has also beenobserved in such ‘hard’ end points as PET, MRI and single-photon emission computed tomography(SPECT)-determined improvement in myocardialperfusion and function in these patients.

These observations indicate that the placebo effect inthese trials is a real biological phenomenon. Part of theexplanation could lie in the improved compliance oftrial patients with their medical regimens. Another partcould be the documented contribution of increasedphysical activity and exercise to the promotion of angio-genesis56. Other, not understood, biological factorsmight also be involved.

The contribution of concomitant drug therapy tothe effectiveness of therapeutic angiogenesis interven-tions has not been thoroughly examined. Anecdotalreports indicate that a number of common medica-tions, including captopril, isosorbide dinitrate, lovas-tatin and furosemide, might potentially interfere withangiogenesis54. Even aspirin and other non-steroidalanti-inflammatory drugs, which are frequently used over-the-counter medications, might have anti-angiogeniceffects57. Equally important could be the anti-angio-genic effects of inhibitors of cyclooxygenase-2 (REF. 58).Careful accounting of concomitant medication usage istherefore required for trials of therapeutic angiogenesis.

SummaryThe field of therapeutic stimulation of new vessel forma-tion has matured and evolved during the past decade. Inthe absence of any clear success stories, all suggestions asto what is likely to work in this new class of drugs mustbe offered tentatively. Indeed, the successful developmentof a drug capable of revascularization of tissues would bea major milestone in the history of medicine. Despitethese challenges, the incentive offered by the large unmetclinical need and the progress in our understanding ofangiogenesis biology lead us to believe that successfulagents will emerge and will open truly revolutionarypossibilities for cardiovascular therapeutics.

A clinical plan to develop an arteriogenic drug forperipheral arterial occlusive disease is attractive, and isin some respects simpler than that for ischaemic heartdisease because of the relative simplicity of intramuscu-lar injection. Patients with peripheral arterial disease canbe grouped into two broad categories: those with severelimb ischaemia at rest, and those with claudication butwithout a critically ischaemic limb. End points for thelatter group include peak walking times and anklebrachial indices, which is a measurement of perfusionand blood pressure of the lower extremity. Limb salvageis a clinically important and clearly determined endpoint in the group with severe limb ischaemia, but, asis the case with coronary artery disease, the complexity ofthe disease process, along with the heterogeneity of thepatient population, makes reducing limb loss a challeng-ing end point to achieve in a double-blinded, placebo-controlled study. In trials that examine the effect ofarteriogenic agents on claudication, some successes havebeen reported; however, the ‘softness’of the peak walkingtime end point has also become evident, as the improvedpeak walking time seen at six months was no longerevident by twelve months5, raising questions as to theclinical significance of the early positive finding.

An important consideration is that the excellentsafety profile of current agents with the current mode ofdelivery could also result from the lack of interactionwith target receptors. For this reason, vigilance for unto-ward effects will need to take the entire clinical deliveryplan into account.

Placebo effect and concomitant medicationsAn important factor that has emerged from early double-blind studies of therapeutic angiogenesis agents is theimportance of the placebo effect in the evaluation of trialdata. Although the occurrence and significance of theplacebo effect has long been appreciated, its extent andprevalence in this patient population surpassed expecta-tions and sets the precedent that the evaluation of effi-cacy is only possible in a double-blind trial format.Strikingly, the placebo effect is not limited to ‘soft’

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Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/angiopoietin-2 | FGF2 | FGF4 | FLT-1 | HIF-1α | MCP-1 | PDGF-B | VEGF

FURTHER INFORMATIONEncyclopedia of Life Sciences: http://www.els.netcardiovascular disease: epidemiology | ischemic heart diseaseAccess to this interactive links box is free online.

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Therapeutic angiogenesis in cardiovascular disease