British Medical Bulletin

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British Medical Bulletin 59:211-225 (2001)
© 2001 Oxford University Press

Angiogenesis, protein and gene delivery

Michael Azrin

Cardiology Division, University of Connecticut Health Center, Farmington, Connecticut, USA

	Abstract

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 
Advances in our understanding of angiogenesis and blood vessel growth have given rise to efforts to develop novel therapeutic approaches for patients with ischaemia who are not adequately treated with presently available therapies. Among the growth factors that play a role in blood vessel growth and development, vascular endothelial growth factors and fibroblast growth factors have been the most extensively studied. Various methods of delivery have been utilized to enhance localization and persistence, including methods for delivery of proteins as well as gene transfer techniques. Initial clinical trials have now been undertaken. Preliminary information on efficacy is beginning to become available, raising hopes, as well as questions about the future direction and potential success of therapeutic angiogenesis as a clinical approach to the treatment of ischaemia.

	Introduction

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 
In spite of significant advances in the treatment of coronary artery disease, a substantial number of patients remain inadequately treated with anti-anginal medications or with coronary revascularization thereby prompting great interest in alternative therapeutic approaches. One approach utilizes the knowledge that the collateral circulation can play an important role in patients with coronary artery disease. Advances in our understanding of the process of angiogenesis, and the recognition that collateral formation can be altered by growth factors have given rise to the concept of therapeutic angiogenesis. This refers to the therapeutic administration of agents such as growth factors or cytokines to enhance collateral development and reduce vascular insufficiency. Demonstrated efficacy in animal studies then led to initial human clinical trials of therapeutic angiogenesis using both protein delivery and gene transfer therapy. Clinical trials are currently being designed and performed, and the future of therapeutic angiogenesis is just beginning to unfold.

The importance of collateral vessels has long been recognized and is often evident in the clinical practice of cardiology. For example, one patient with an occluded left anterior descending (LAD) coronary artery and inadequate collaterals might present with an acute myocardial infarction and severe left ventricular dysfunction, while another patient, who also has an occluded LAD, but in whom the only evident difference is the presence of abundant collaterals, may have only angina and preserved left ventricular function. The importance of the collateral circulation has been studied systematically, and the ability of collateral circulation to limit ischaemia has been demonstrated prospectively¹. During acute coronary occlusion, a gradient develops between a patent vessel and the occluded coronary resulting in the recruitment or filling of dormant collateral vessels; this has been demonstrated in the setting of acute coronary occlusion such as occurs during coronary spasm or during balloon angioplasty². Furthermore, the presence of collateral vessels has been demonstrated to modify the functional effects of coronary occlusion by reducing ST changes, metabolic effects and infarct size, and to improve ejection fraction and long-term outcomes³. Thus, it is evident that collaterals can play an important functional role.

In addition to recruitment of pre-existing collaterals, collateral flow may be enhanced by both enlargement of existing channels, as well as by the development of new collateral channels⁴. The former is arteriogenesis, which refers to the remodelling and structural enlargement of pre-existing arteriolar connections, and the latter is angiogenesis which refers to extension of the existing vasculature through the development of capillary sprouts (a process that also occurs in the setting of tumour neovascularization, wound healing and pathological diabetic retinopathy). Angiogenesis is a complex multistep process. In response to an angiogenic stimulus, the normally quiescent endothelial cells become activated. Local vasodilation, increased permeability and proteolytic disruption of the basement membrane then lead to migration of endothelial cells through the basement membrane into the surrounding matrix with proliferation and formation of capillary sprouts. Formation of a lumen and connection to other vascular structures with reformation of the basement membrane results in a functional vascular connection.

Several factors can influence collateral development. Ischaemia is felt to be a strong stimulant. For example, enhanced collaterals have been demonstrated in dogs subjected to long-term hypoxia or with chronic anaemia and in animal models of coronary or limb ischaemia. Similarly, humans with chronic anaemia or chronic obstructive pulmonary disease have more extensive collaterals, and extensive collateralization can be observed in the setting of chronic coronary disease or claudication. But, collateral development is also under the influence of factors other than ischaemia. For example, patients with distal lower extremity ischaemia will often develop extensive collaterals proximally, in the thigh, in an area that is not itself ischaemic. Increased shear forces develop due to the pressure differential caused by vessel occlusion. The stimuli for collateral development include these increased shear forces which result in endothelial activation, as well as an important contribution by monocytes. While both small vessel collaterals arising from capillaries and post-capillary venules, and larger visible collaterals arising from pre-existing interconnecting arterioles both may contribute to tissue perfusion in the setting of vessel occlusion, the larger collateral vessels are felt to contribute more to meaningful perfusion⁵.

Regulation of angiogenesis and collateral formation has been demonstrated to be complex, involving stimulators, inhibitors and modulators. While a number of cytokines play important roles, two families of growth factors which play key roles (and that have been the most extensively investigated and utilized in clinical and preclinical studies) are the vascular endothelial growth factor (VEGF) and the fibroblast growth factor (FGF) protein families.

	Vascular endothelial growth factor (VEGF)

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 
VEGF is a key factor in the process of angiogenesis⁶. It is secreted by a number of cell types and acts in a paracrine fashion. VEGF exists as a number of isoforms that are produced by alternative splicing resulting in isoforms of 121, 145, 165, 189 or 206 amino acids. VEGF also contains a typical signal sequence enabling secretion by intact cells. The 165 and 121 residue forms are the most abundantly expressed. The larger isoforms contain the binding sites for heparan sulphate and extracellular matrix; therefore, a significant portion of the larger isoforms remains bound to the cell surface or extracellular matrix. In contrast, VEGF₁₂₁ does not contain the heparin- or matrix-binding domains making it the most freely diffusible form.

VEGF is a highly specific mitogen for vascular endothelial cells. In vitro, VEGF induces endothelial cell proliferation and chemotaxis. VEGF also induces vascular hyperpermeability allowing small molecules to pass into the extravascular space. There are two well-described VEGF receptors: FLT-1 (VEGFR-1) and KDR (FLK-1, VEGFR-2) which are found almost exclusively on endothelial cells. FLT-1 (VEGFR-1) appears to be involved in vessel permeability, while KDR (VEGFR-2) mediates the angiogenic process.

The VEGF protein family includes VEGF (also called VEGF-A) as well as VEGF-B, VEGF-C (VEGF-2), VEGF-D and placental growth factor. While VEGF has been investigated the most extensively, both VEGF-2 and VEGF-D have also been studied for therapeutic angiogenesis, in part because, like VEGF, they too interact with the KDR (VEGFR-2) receptor involved in angiogenesis.

VEGF plays a key role in angiogenesis and has effects on all of the key steps in this process, including endothelial cell mitogenesis and chemotaxis, vascular permeability, and proteolysis. Both VEGF and its endothelial specific receptors are up-regulated in the setting of hypoxia – potentially enabling localization of the effect to areas of hypoxia or ischaemia⁷. These key characteristics have made VEGF an appealing candidate for use in therapeutic angiogenesis.

	Fibroblast growth factor (FGF)

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 
The fibroblast growth factor family includes more than 20 different structurally-related polypeptides characterized by high affinity binding to cellular heparan sulphates. The first two members of the fibroblast growth factor family identified, FGF-1 (acidic FGF) and FGF-2 (basic FGF), have been extensively studied and have significant effects on the processes involved in angiogenesis⁸. In spite of the historical name of ‘fibroblast’ growth factors, they are in fact potent mitogens for numerous cell types including endothelial cells, smooth muscle cells and cells of mesenchymal, neural and epithelial origin. FGFs regulate the key migratory and proliferative processes involved in angiogenesis. The prototypic fibroblast growth factors, FGF-1 and FGF-2, are chemotactic and mitogenic for endothelial cells, they induce matrix-resorbing activity and proteolytic systems that are necessary for matrix degradation involved in angiogenesis, and they are powerful angiogenic agents both in vitro and in vivo⁹.

	Methods of delivery

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 
Therapeutic angiogenesis represents an attempt to induce blood vessel growth in an area of ischaemia while avoiding unwanted angiogenesis elsewhere. While some degree of localization might be expected through the up-regulation of receptors for angiogenic growth factors in regions of ischaemia¹⁰, there remains a need to localize the effects of therapy to avoid such potential side-effects as unwanted angiogenesis, mitogenesis or growth factor-mediated hypotension, as well as to deliver a sufficient quantity of therapy locally for a sufficient duration. Two different approaches have been taken to achieve localization and persistence. The first is to localize physically the protein (or the gene) to the region of interest; the second is to utilize gene therapy to achieve more prolonged local expression.

Techniques for localization of drug delivery have been developed, and several have been utilized in clinical or preclinical trials of therapeutic angiogenesis. The simplest delivery techniques have included intravenous, intra-arterial, or peripheral intramuscular injection. Although the delivery techniques required are more involved, angiogenic growth factors have also been delivered directly to the myocardium, either as epicardial injections at the time of surgery or through catheter-based endomyocardial injection techniques. In addition, perivascular delivery has been employed either by peri-adventitial delivery using sustained-release polymers at the time of surgery or by intrapericardial delivery. All of these techniques have the same goal which is to enhance the local effect while avoiding systemic effects. Each of these techniques has apparent benefits and drawbacks¹¹. The relative ease of delivery by methods such as intracoronary or intravenous delivery, may result in more systemic exposure, and will have to be compared in terms of safety, efficacy and clinical applicability to techniques designed to localize delivery.

	Gene therapy

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 
Gene therapy for angiogenesis involves transfer of the gene encoding the angiogenic protein into a host cell to obtain over-expression of the protein. Several different means of gene transfer have been considered, including both viral and non-viral methods. Viral vectors have been employed because viruses have evolved highly efficient mechanisms to transfer their genetic material to target cells. Viral gene delivery involves the incorporation of the chosen DNA sequences into the genome of the parent virus, which then transfers the gene to the target cells.

Viral-based gene therapy

Retroviral vectors have been used extensively for gene transfer. However, their use in clinical angiogenesis trials has several limitations. Retroviral (RNA) vectors integrate into the host genome, resulting in permanent expression of the therapeutic gene. This is in contrast to adenoviral (DNA) vectors which remain independent of the host genome and are gradually lost with cell division. Retroviral vectors are truly defective and none of the viral proteins are expressed in the target cell, thereby avoiding an immune response. However, retroviruses are small, limiting the size of genes that can be incorporated. In addition, transfer efficiency is low, and they are not infective in non-dividing cells, which is a major limitation for this application. Also, because retroviral vectors integrate into the host cell chromosome, insertional mutagenesis is possible.

Adenoviral vectors have become widely used for experimental gene transfer. They can infect non-dividing cells, do not integrate into the host genome and have a high efficiency of transfer. The production of ‘replication-deficient’ vectors results in a reduced host immune response, and is also important for patient safety, as well as to prevent transfer to other individuals. For adenoviral vectors, the approach taken was to remove critical viral genes necessary for viral propagation. The gene for the angiogenic growth factor is then inserted along with a promoter, such as the CMV promoter, which can then drive constitutive expression of the inserted protein gene. The duration of effect is limited to only a few weeks because cells derived from the modified cells will not express the transgene, and because genes expressed by adenoviral vectors elicit an immune response. Early non-specific inflammation may occur, and this is followed by a specific host immune response resulting in a shortened duration of transgene expression. While transient expression is a limitation of therapy for genetic disorders, it may actually be a beneficial safety attribute in the treatment of ischaemia where indefinite growth factor expression may not be required or desirable.

Non-viral gene therapy

One of the simplest means of delivery of a gene to a target cell is through the use of ‘naked’ or plasmid DNA. Plasmid DNA expression vectors are comprised of DNA sequences in addition to the gene of interest. These include a transcriptional promoter, as well as sequences that impart stability and functionality to the mRNA.

When plasmid DNA is placed in contact with the cell membrane, a small amount will pass into the cell. This non-viral means of delivery of genes to target cells has a number of potential advantages. Several of the safety concerns associated with viral vectors, including an immune response, insertional mutagenesis or viral transmission, may be avoided. However, plasmid-based gene therapy must overcome several obstacles including penetration of the cell plasma membrane, passage through the cytoplasm, entry into the nucleus, transcription of DNA into functional mRNA, transport of the mRNA to the cytoplasm, and translation into a functional protein. While viruses have evolved to transfer efficiently their nucleic acid to mammalian cells, plasmid gene transfer remains inefficient. In spite of this, two factors may enable therapeutic angiogenesis using plasmid gene therapy. First, there is evidence that gene transfer is enhanced in ischaemic muscle¹². Second, use of a secreted protein may have paracrine effects in spite of low transfection efficiency¹³. Furthermore, successful gene transfer for therapeutic angiogenesis has been achieved in animals as well as in preliminary clinical trials using plasmid VEGF¹⁴.

	Clinical trials

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 
A number of clinical trials have now been performed (Table 1). While most of these are small trials, they have provided preliminary information regarding safety, feasibility and dosing which is now leading to larger clinical trials to evaluate efficacy. A number of approaches have been undertaken. These include use of proteins, plasmids or viral-based gene transfer therapy, use of both VEGF and FGFs, as well as various delivery approaches including intravenous, intra-arterial, intramuscular, perivascular and therapy administered in concert with revascularization.

View this table: [in this window] [in a new window]   Table 1 Clinical therapeutic angiogenesis trials

 
VEGF protein therapy

A series of studies has been performed which ultimately led to the use of VEGF protein in clinical trials of therapeutic angiogenesis. First, it was recognized that VEGF plays a key role in a number of settings, ranging from vascular embryogenesis to pathological angiogenesis. Angiogenic properties were demonstrated in experimental angiogenesis models such as the chick chorio-allantoic membrane and the rabbit cornea, and then in ischaemic animal models including the rabbit hindlimb model and canine and porcine coronary ischaemia models. In addition, various routes of administration were utilized including intravenous, intra-arterial, peri-adventitial and intramyocardial routes in animal models. These studies, which have been reviewed elsewhere^6,15 provided the basis for initiating clinical trials of VEGF.

Phase I trials were performed to evaluate safety and tolerability of both intracoronary¹⁶ and intravenous administration¹⁷. These studies were performed on patients with demonstrable myocardial ischaemia who were not candidates for revascularization by surgery or by percutaneous coronary intervention. VEGF₁₆₅ protein was administered to 15 patients by intracoronary infusion in escalating doses. Hypotension was found to be dose-limiting with a maximal tolerated dose of 50 ng/kg/min. While there was no placebo group, there was nevertheless a suggestion of improvement clinically and by functional testing. Of the 15 patients, 13 had a decrease in angina class; 7 of 14 patients had an improvement in myocardial perfusion imaging on the rest studies, though not in the stress studies, and each of those 7 patients had a corresponding increase in angiographic collateral density¹⁶. In a second phase I trial¹⁷, VEGF was administered intravenously to 28 patients. The maximal tolerated dose was again 50 ng/kg/min, and there were again suggestions of improvement in rest myocardial perfusion (which improved in two or more segments in 54% of patients) and in collateral density in this small dose-finding study.

With these dose finding studies as background, the VIVA trial (VEGF in Ischemia for Vascular Angiogenesis) was then performed¹⁸. This was a phase II double-blind placebo-controlled trial of VEGF₁₆₅ by intracoronary (17 or 50 ng/kg/min) followed by intravenous administration of the same dose on days 3, 6 and 9. VEGF was well tolerated with no significant increase in adverse events. The primary end-point was exercise treadmill time at day 60. There was a notable improvement in all three groups, including the placebo group, at 60 days, in exercise time, angina class, as well as quality of life compared to baseline values. However, compared with the placebo group, there was no improvement in the primary end-point. While these early results were disappointing, subsequent findings suggest that the placebo effect, which was prominent at 60 days, subsequently diminished, whereas the improvements in the high dose group were maintained or increased^19,20. At 120 days, there was improvement in angina class in the high dose group in comparison with the placebo group (P = 0.04), and a trend for improvement in exercise time (P = 0.17). While this trial was not a definitive phase 3 trial of efficacy, it was nevertheless important because of the larger number of patients enrolled and because it was double-blind, randomized and placebo-controlled. It demonstrated the critical importance of careful placebo controls in studies of angiogenesis The explanation for the absence of clear benefit may lie in methodological issues, the marked placebo effect, a more delayed time-course than could be identified in this short-term study, or simply may reflect an inadequate effect on angiogenesis and collateral development.

VEGF plasmid DNA therapy

Clinical trials of VEGF using direct injection of plasmid VEGF DNA were stimulated by a series of illustrative animal studies. After demonstration of the angiogenic potential of VEGF protein in preclinical studies, similar studies were performed using plasmid DNA. An initial animal study utilized specialized catheters to achieve localized, intra-arterial delivery²¹. However, subsequent studies demonstrated that intramuscular injection, which is potentially simpler to achieve and does not require arterial cannulation, could also result in functional effects in spite of low efficiency of transduction²².

Clinical trials were performed in patients with peripheral vascular disease using catheter-based direct intra-arterial delivery including a dose finding study in 8 patients using Plasmid VEGF₁₆₅ delivered with the hydrogel-coated balloon²³. Although there were no placebo controls, there were improvements in symptoms and blood flow at the high dose, as well as a transient telangiectasia and peripheral oedema in one patient at the highest dose²⁴. Similarly, in the coronary circulation catheter-based techniques for local gene delivery using a perfusion catheter were utilized to deliver plasmid VEGF/liposome which demonstrated feasibility and safety, although efficacy of therapeutic angiogenesis was not evaluated²⁵.

The demonstration of successful skeletal muscle VEGF gene transfer in animal studies suggested that intra-arterial delivery might not be required and led to clinical trials of intramuscular injection of plasmid VEGF₁₆₅ in patients with peripheral vascular disease in a total of 50 patients²⁶. Preliminary reports describing 9 patients with critical limb ischaemia¹⁴ and 6 patients with Buerger's disease²⁷ both demonstrated transgene expression. In addition, some patients in these studies, which did not include placebo controls, did have functional improvement including improved blood flow, healing of ulcers and improved symptoms, particularly in patients with Buerger's disease. These preliminary feasibility studies, as well as a promising preliminary report of 13 patients receiving plasmid VEGF-2 in a placebo-controlled trial²⁸, have given rise to the initiation of clinical trials with intramuscular plasmid DNA injection.

The demonstration of possible clinical benefits with intramuscular injection for peripheral vascular disease also led to trials of intramyocardial VEGF gene transfer. Initially, intramyocardial injection could only be achieved intra-operatively either at the time of bypass surgery or through direct injection through a thoracotomy. A series of 30 patients were treated with intramyocardial plasmid VEGF₁₆₅ as sole therapy (i.e. without concomitant CABG) in a dose escalation trial^29–32. Serum VEGF levels increased, confirming successful gene transfer. There were also improvements in angina, exercise time, and nuclear perfusion studies compared to baseline.

Studies involving surgical intramyocardial injections are limited by the morbidity of concomitant surgery, the inability to enroll patients in a placebo arm of a trial of surgical therapy, and the confounding effects of concomitant bypass grafting in combination trials. The development of percutaneous techniques for intramyocardial injection has enabled the initiation of placebo-controlled trials for coronary ischaemia and also may enable repeat dosing³¹.

VEGF adenoviral vector gene therapy

In addition to trials of VEGF protein, plasmid VEGF₁₆₅ or VEGF-2³³, trials of gene transfer using an adenoviral vector are also underway. An adenoviral VEGF₁₂₁ vector was utilized in a dose escalation trial in 21 patients (15 patients underwent concomitant coronary bypass grafting, and 6 patients received sole therapy with adenoviral VEGF₁₂₁ through a mini-thoracotomy or thorocoscopy), further demonstrating the feasibility of intramyocardial angiogenic therapy^34,35. There was no evident cardiac or systemic toxicity. In the sole therapy arm, all patients had improved angina, and there were trends for improvement in angiographic collaterals, stress wall motion, reversible ischaemia and exercise parameters. A randomized, double-blind, placebo-controlled phase 2 trial of intramuscular adenoviral VEGF₁₂₁ is now underway in patients with claudication.

FGF protein therapy for myocardial ischaemia

FGF-2 and to a lesser extent FGF-1 have been extensively studied in animal models of both peripheral and myocardial ischaemia. Several different routes of administration including intra-arterial, intravenous, intramuscular, perivascular and subcutaneous routes have been utilized. Reviews of the preclinical data have been published⁹ and, based on promising preclinical data in animal models of ischaemia, clinical trials of both protein and gene therapy with FGFs have been undertaken.

Schumacher and colleagues^36,37 published the first placebo-controlled therapeutic angiogenesis trial. They administered FGF-1 protein at the time of bypass surgery as an intramyocardial injection distal to the site of the LAD anastomosis in 40 patients undergoing LIMA grafting of the LAD where an additional stenosis was present in the distal LAD or one of the diagonals. Initial evaluation by digital subtraction angiography at 3 months demonstrated a significant increase in capillary density, as well as collateral filling of non-bypassed vessels in the treated patients, but not in the control patients. While the initial report did not evaluate the functional benefit of this therapy, subsequent follow-up at 3 years in 31 of the original 40 patients demonstrated an improvement in ejection fraction and NYHA functional class.

FGF-2 has also been utilized as an adjunct to bypass surgery. Sellke and colleagues³⁸ performed a small study in 8 patients in whom FGF-2 was administered epicardially in a slow-release formulation within heparin alginate beads placed epicardially at the time of CABG in areas of incomplete revascularization. In this study, as well as in an expanded series³⁹ of 24 patients (which included 8 controls) there was evidence of improved perfusion by magnetic resonance imaging as well as by nuclear perfusion imaging, particularly at the highest dose. These studies are nevertheless limited by the confounding effect of the concomitant bypass surgery as well as the small number of patients enrolled.

FGF-2 protein has also been delivered as an intracoronary infusion. Unger and colleagues⁴⁰ performed a phase 1 study of intracoronary FGF-2 infusion in 25 patients with stable angina which included 8 placebo-treated patients. Hypotension, bradycardia, and transient mild thrombocytopenia and proteinuria were noted in this dose ranging study. A larger phase I dose-finding study by Laham and colleagues⁴¹,in 52 patients with ischaemia who were not amenable to revascularization, demonstrated that intracoronary FGF-2 was safe and well-tolerated up to a dose of 36 µg/kg. While this was a patient population at high risk for cardiac and other adverse events, there was also dose-related hypotension and proteinuria in 4 patients. Follow-up evaluation demonstrated improvements in angina, measures of quality of life, exercise tolerance and ejection fraction, as well as a reduction in the extent of ischaemia by magnetic resonance imaging. Nuclear perfusion studies also suggested improvement after FGF-2 in some measures including a reduction in segmental reversible ischaemia at 29, 57 and 180 days, and improved rest perfusion in patients that had areas of mild or moderate (but not severe) resting hypoperfusion⁴². While the dose ranging nature of these trials, the small size and the lack of a blinded placebo group limited the strength of any conclusions about efficacy, they nevertheless provided the groundwork for a subsequent larger trial.

Preliminary results of a phase II randomized, double-blind, placebo-controlled trial of intracoronary FGF-2 (the FIRST study) have recently been presented⁴³. In this trial, 337 patients with angina, who were ineligible for angioplasty or CABG were randomized to receive either placebo or one of three doses of FGF-2 (0.3, 3.0 or 30 mcg/kg) by a single intracoronary infusion. The primary end-point was exercise capacity evaluated at 90 days. There was a 65 s improvement in patients receiving one of the FGF doses; however, there was a 45 s improvement in the placebo group and this difference was not significant. In subgroups, including older patients and patients with more baseline angina, there were significant differences in exercise time as well as in angina class Nevertheless, in spite of promising preclinical and preliminary clinical data, the FIRST trial, like the VIVA trial of VEGF which also utilized recombinant protein, demonstrated a significant placebo effect and no significant difference in the primary endpoint.

FGF protein therapy for peripheral vascular disease

FGF-2 has also been administered to patients with peripheral vascular disease. Lazarous and colleagues⁴⁴ performed a phase 1 double-blind, placebo-controlled trial in 19 patients with claudication in which FGF-2 was administered as an intra-arterial infusion. They reported significantly increased blood flow assessed by plethysmography, compared with baseline blood flow, as well as a decrease in claudication at the highest dose of 30 µg/kg.

Preliminary results of a larger double-blind, placebo-controlled trial in patients with claudication have recently been presented⁴⁵. In the TRAFFIC trial (therapeutic angiogenesis with FGF-2 for intermittent claudication), 190 patients with claudication were randomized to receive either one or two doses of intra-arterial FGF-2 protein (30 µg/kg) given 30 days apart. The primary end-point was the change in peak walking time at day 90. There was an acceptable safety profile including a low incidence of hypotension and proteinuria. At 90 days, there was a 14% increase in the peak walking time in the placebo group, while there was a 34% increase in the peak walking time in the single dose group, (P = 0.026) and a 20% increase in the double-dose group (P = NS). This clinical improvement in the single-dose group was supported by measurement of ankle-brachial index, and by improvement in claudication severity. Although not a definitive phase 3 trial, this trial of intra-arterial protein therapy for claudication is the first phase 2 therapeutic angiogenesis trial to demonstrate a positive result in the primary endpoint.

	Conclusions

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 
The identification of potent angiogenic proteins, the promising preclinical data and the development of methods of delivery suggested that therapeutic angiogenesis might be achievable. Approaches that were tested in preclinical studies have now been evaluated in small and often uncontrolled human feasibility studies. The limited clinical data now available contain evidence suggesting that functional benefit may be obtained in patients with symptomatic ischaemia. While the two phase 2 trials, the VIVA trial and the FIRST trial, did not demonstrate significant improvement in their primary end-points, a third, the TRAFFIC, trial has demonstrated improvement with one of two dosing regimens. In addition, suggestive, albeit not universally significant, benefits were seen in all three, and the safety data have been reassuring. Nevertheless, the number of questions that remain is substantial. Which patients should be treated? Which of the ever increasing number of angiogenic growth factors should be used? Or, will it require combinations of growth factors? Which method of delivery and which dosing regimen is best? Will functional collaterals be formed, or merely non-functioning capillary networks? Can exogenously administered growth factors or even gene transfer therapies succeed in a complex clinical setting where the adaptive responses to ischaemia have failed? There is certainly reason for optimism given the encouraging preliminary data, but there is also ample room for scepticism. The ideal angiogenic agent will be simple to administer, will be effective, and will have limited side effects. However, clinical trials that convincingly demonstrate the efficacy of any single approach are needed, before the differing approaches can be compared.

	Footnotes

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 
Correspondence to:Michael Azrin MD, Cardiology Division, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030, USA

	References

Top Footnotes Abstract Introduction Vascular endothelial growth... Fibroblast growth factor (FGF) Methods of delivery Gene therapy Clinical trials Conclusions References

 

1. Charney R, Cohen M. The role of the coronary collateral circulation in limiting myocardial ischemia and infarct size. Am Heart J 1993; 126: 937–45[Web of Science][Medline]
2. Rentrop KP, Cohen M, Blanke H, Phillips RA. Changes in collateral channel filling immediately after controlled coronary artery occlusion by an angioplasty balloon in human subjects. J Am Coll Cardiol 1985; 5: 587[Abstract]
3. Goncalves LM. Angiogenic growth factors: potential new treatment for acute myocardial infarction. Cardiovasc Res 2000; 45: 294–302[Web of Science][Medline]
4. Schaper W, Ito WD. Molecular mechanisms of coronary collateral vessel growth. Circ Res 1996; 79: 911–9[Free Full Text]
5. Schaper W, Schaper J. Collateral Circulation. Norwell, MA: Kluwer, 1993
6. Ferrara N. Vascular endothelial growth factor: molecular and biological aspects. Curr Top Microbiol Immunol 1999; 237: 1–30[Web of Science][Medline]
7. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359: 843–5[Medline]
8. Slavin J. Fibroblast growth factors: at the heart of angiogenesis. Cell Biol Int 1995; 19: 431–44[Web of Science][Medline]
9. Simons M, Laham RJ. Therapeutic angiogenesis in myocardial ischemia. In: Ware JA, Simons M. (eds) Angiogenesis and Cardiovascular Disease. New York: Oxford University Press, 1999
10. Sellke FW, Wang SY, Stamler A et al. Enhanced microvascular relaxations to VEGF and bFGF in chronically ischemic porcine myocardium. Am J Physiol 1996; 271: H713–20[Web of Science][Medline]
11. Kornowski R, Fuchs S, Leon MB, Epstein SE. Delivery strategies to achieve therapeutic myocardial angiogenesis. Circulation 2000; 101: 454–8[Abstract/Free Full Text]
12. Takeshita S, Ishiki T, Sato T. Increased expression of direct gene transfer into skeletal muscles observed after acute ischemic injury in rats. Lab Invest 1996; 74: 1061–5[Web of Science][Medline]
13. Losordo DW, Pickering JG, Takeshita S et al. Use of the rabbit ear artery to serially assess foreign protein secretion after site specific arterial gene transfer in vivo: evidence that anatomic identification of successful gene transfer may underestimate the potential magnitude of transgene expression. Circulation 1994; 89: 785–92[Abstract/Free Full Text]
14. Baumgartner I, Pieczek A, Manor O et al Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 1998; 97: 1114–23[Abstract/Free Full Text]
15. Henry TD, Abraham JA. Review of preclinical and clinical results with vascular endothelial growth factors for therapeutic angiogenesis. Curr Intervent Cardiol 2000; 2: 228–41
16. Hendel RC, Henry TD, Rocha-Singh K et al. Effect of intracoronary recombinant human vascular endothelial growth factor (rhVEGF) on myocardial perfusion: evidence for a dose-dependent effect. Circulation 2000; 101: 118–21[Abstract/Free Full Text]
17. Gibson CM, Laham R, Giordano FJ et al. Magnitude and location of new angiographically apparent coronary collaterals following intravenous VEGF administration. J Am Coll Cardiol 1999; 33: 384A
18. Henry T, Annex B, Azrin M et al. Double blind placebo controlled trial of recombinant human vascular endothelial growth factor – the VIVA trial. J Am Coll Cardiol 1999; 33: 384A
19. Henry TD, Annex BH, Azrin MA et al. Final results of the VIVA trial of rhVEGF for human therapeutic angiogenesis. Circulation 1999; 100: I-476
20. Henry T, McKendall GR, Azrin MA et al. Viva trial: one year follow up. Circulation 2000; 102: II-129
21. Riessen R, Rahimizadeh H, Blessing B et al. Arterial gene transfer using pure DNA applied directly to a hydrogel-coated angioplasty balloon. Hum Gene Ther 1993; 4: 749–58[Web of Science][Medline]
22. Takeshita S, Weir L, Chen D et al. Therapeutic angiogenesis following arterial gene transfer of vascular endothelial growth factor in a rabbit model of hindlimb ischemia. Biochem Biophys Res Commun 1996; 227: 628–35[Web of Science][Medline]
23. Isner JM. Arterial gene transfer of naked DNA for therapeutic angiogenesis: early clinical results. Adv Drug Delivery Rev 1998; 30: 185–97[Web of Science][Medline]
24. Isner JM, Pieczek A, Schainfeld R et al. Clinical evidence of angiogenesis following arterial gene transfer of phVEGF165 in patient with ischemic limb. Lancet 1998; 348: 370–4
25. Laitinen M, Hartikainen J, Hiltunen MO et al. Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty. Hum Gene Ther 2000; 11: 263–70[Web of Science][Medline]
26. Baumgartner I, Rauh G, Pieczek A et al. Lower-extremity edema associated with gene transfer of naked DNA encoding vascular endothelial growth factor. Ann Intern Med 2000; 132: 880–4[Abstract/Free Full Text]
27. Isner JM, Baumgartner I, Rauh G et al. Treatment of thromboangitis obliterans (Buerger's disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results. J Vasc Surg 1998; 28: 964–73[Web of Science][Medline]
28. Rauh G, Gravereaux E, Pieczek A et al. Assessment of safety and efficiency of intramuscular gene therapy VEGF-2 in patients with critical limb ischemia. Circulation 1999; 100: I-770
29. Losordo DW, Vale PR, Symes JF et al. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. Circulation 1998; 98: 2800–4[Abstract/Free Full Text]
30. Symes JF, Losordo DW, Vale PR et al. Gene therapy with vascular endothelial growth factor for inoperable coronary artery disease. Ann Thorac Surg 1999; 68: 830–7[Abstract/Free Full Text]
31. Vale PR, Losordo DW, Milliken CE et al. Left ventricular electromechanical mapping to assess efficacy of phVEGF165 gene transfer for therapeutic angiogenesis in chronic myocardial ischemia. Circulation 2000; 102: 965–74[Abstract/Free Full Text]
32. Lathi KG, Vale PR, Losordo DW et al. Gene therapy with vascular endothelial growth factor for inoperable coronary artery disease: anesthetic management and results. Anesth Analg 2001; 92: 19–25[Abstract/Free Full Text]
33. Hendel RC, Vale PR, Losordo DW et al. The effects of VEGF-2 gene therapy on rest and stress myocardial perfusion: results of serial SPECT imaging. Circulation 2000; 102: II-769
34. Rosengart TK. Six-month assessment of a phase I trial of angiogenic gene therapy for the treatment of coronary artery disease using direct intramyocardial administration of an adenovirus vector expressing the VEGF₁₂₁ cDNA. Ann Surg 1999; 230: 466–70[Web of Science][Medline]
35. Rosengart TK, Lee LY, Patel SR et al. Angiogenesis gene therapy: phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF₁₂₁ cDNA to individuals with clinically significant severe coronary artery disease. Circulation 1999; 100: 468–74[Abstract/Free Full Text]
36. Schumacher B, Pecher P, von Specht BU et al. Induction of neoangiogenesis in ischemic myocardium by human growth factors. Circulation 1998; 97: 645–50[Abstract/Free Full Text]
37. Pecher P, Schumacher BA. Angiogenesis in ischemic human myocardium: clinical results after 3 years. Ann Thorac Surg 2000; 69: 1414–9[Abstract/Free Full Text]
38. Selke FW, Laham RJ, Edelman ER et al. Therapeutic angiogenesis with basic fibroblast growth factor. Ann Thorac Surg 1998; 65: 1540–4[Abstract/Free Full Text]
39. Laham RJ, Selke FW, Edelman ER et al. Local perivascular delivery of basic fibroblast growth factor in patients undergoing coronary bypass surgery. Circulation 1999; 100: 1865–71[Abstract/Free Full Text]
40. Unger EF, Goncalves L, Epstein SE et al. Effects of a single intracoronary injection of basic fibroblast growth factor in stable angina pectoris. Am J Cardiol 2000; 85: 1414–9[Web of Science][Medline]
41. Laham RJ, Chronos NA, Pike M et al. Intracoronary basic fibroblast growth factor (FGF-2) in patients with severe ischemic heart disease: results of a phase I open-label dose escalation study. J Am Coll Cardiol 2000; 36: 2132–9[Abstract/Free Full Text]
42. Udelson JE, Dilsizian V, Laham RJ et al. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 improves stress and rest myocardial perfusion abnormalities in patients with severe symptomatic chronic coronary artery disease. Circulation 2000; 102: 1605–10[Abstract/Free Full Text]
43. Chronos N. FIRST, FGF-2 Initiating Revascularization Support Trial. Meeting of the American College of Cardiology, Anaheim, CA, USA, March 2000
44. Lazarous DF, Unger EF, Epstein SE et al. Basic fibroblast growth factor in patients with intermittent claudication: results of a phase I trial. J Am Coll Cardiol 2000; 36: 1239–44[Abstract/Free Full Text]
45. Lederman R. American Heart Association Scientific Conference on Therapeutic Angiogenesis and Myocardial Laser Revascularization, Santa Fe, NM, USA, January 2001

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