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THE FUTURE OF THERAPEUTIC MYOCARDIAL ANGIOGENESIS

Boodhwani, Munir*; Sodha, Neel R.*; Laham, Roger J.; Sellke, Frank W.*

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doi: 10.1097/01.shk.0000225318.08681.a7
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Abstract

Cardiovascular disease is not only the leading cause of death, disability, and health care expenditure in the United States but also the leading cause of mortality around the world. In a large number of patients, coronary artery disease (CAD) can be of such a diffuse and severe nature that repeated attempts at catheter-based interventions and surgical bypass may be unsuccessful at restoring normal myocardial blood flow. Up to 20% to 37% of patients with ischemic heart disease either cannot undergo coronary artery bypass grafting (CABG) or percutaneous coronary intervention or receive incomplete revascularization with these standard revascularization strategies (1-5). Furthermore, incomplete revascularization has been associated with increased mortality and poorer clinical outcome (6, 7).

Contrary to conventional revascularization techniques that attempt to increase blood flow to ischemic myocardium through interventions on epicardial coronary vessels, the goal of therapeutic angiogenesis is to restore perfusion by inducing new blood vessel formation, typically through the use of angiogenic growth factors. Early experiments in myocardial angiogenesis using recombinant growth factors or gene-based delivery led to great enthusiasm about their therapeutic potential. However, subsequent application in phase I to III clinical studies has demonstrated limited clinical benefit, and growth factor therapy remains an experimental treatment for patients who have failed conventional therapies.

The discordance between successful preclinical studies and disappointing clinical trials may be explained by a number of factors (8). First, angiogenesis is a complex process that involves interactions between a number of proangiogenic and antiangiogenic mediators, the endothelium, and the extracellular matrix. It is therefore not surprising that single-agent growth factor therapy has not led to large functional improvements in patients. Second, patients with end-stage coronary disease are vastly different from the young and healthy animals in which preclinical testing is typically conducted. The presence of diabetes, hypercholesterolemia, and endothelial dysfunction can significantly limit the effect of growth factors on the angiogenic response (9, 10). Third, the optimal delivery strategy, one that provides local delivery and prolonged exposure to a sufficient dose of growth factor without causing unwanted effects, remains to be discovered. Lastly, the lack of sensitive assays of myocardial angiogenesis limits our ability to detect small subclinical changes that may be occurring in response to growth factor delivery. Despite these limitations, angiogenesis is a critical process that occurs in all humans and, if appropriately modulated, can provide therapeutic benefit to the large population of patients with ischemic CAD.

PROCESSES INVOLVED IN BLOOD VESSEL FORMATION

Vasculogenesis, angiogenesis, and arteriogenesis are three processes that may contribute to the growth of blood vessels (11). As seen in embryonic development, vasculogenesis is the formation of new vessels from pluripotent stem cells. Increasing evidence suggests vasculogenesis may also occur in the adult, as seen in the mobilization of endothelial progenitor cells from bone marrow and the incorporation of these cells into foci of neovascularization. Angiogenesis refers to the growth of capillaries from enlarged venules that sprout capillary buds, become divided by periendothelial cells (intussusception), or are separated by transendothelial cell bridges (bridging) to form capillaries. The process starts with vasodilation and increased permeability to allow extravasation of proteins that modify the extracellular matrix. This is followed by endothelial cell proliferation, migration, and tube formation with endothelial cell differentiation in response to the local tissue environment. Angiogenesis is the manner by which capillaries proliferate in healing wounds, along the border of myocardial infarctions as well as in neoplasms. Whether these vessels are capable of producing physiologically relevant increases in tissue perfusion is debated. Arteriogenesis is the process that results in the appearance of arteries possessing a fully developed tunica media by proliferation of preexisting arterioles into true collateral arteries. Smooth muscle cells may differentiate from various cell types, including endothelial cells and bone marrow precursors. Arteriogenesis involves smooth muscle cell growth and proliferation, migration, and differentiation to a contractile phenotype (12). An example of arteriogenesis is the development of angiographically visible collaterals in patients with advanced obstructive coronary or peripheral vascular disease (Fig. 1).

Fig. 1
Fig. 1:
Coronary angiography in two patients who had an asymptomatic occlusion of the right coronary artery with extensive collaterals from the left coronary system. The right coronary artery (black arrows) fills by intramyocardial collaterals (left, white arrows) or large-bore epicardial collaterals (right, white arrows) underscoring the native collateralization process.

GROWTH FACTORS FOR MYOCARDIAL ANGIOGENESIS

Angiogenesis involves a complex molecular signaling cascade. A significant number of cytokines involved in this process have been identified, including members of the fibroblast growth factor (FGF) family, vascular endothelial growth factor (VEGF) family, platelet-derived growth factor family, and angiopoietins (13). VEGFs and FGFs are the most widely studied and used for clinical studies and will serve as the basis for this discussion.

Vascular endothelial growth factors

VEGFs are a family of heparin-binding glycoproteins shown to act as mitogens for vascular endothelial cells and stimulate endothelial progenitor cell mobilization from the bone marrow (14). The family of VEGF molecules includes VEGF (A-D) and placental growth factor. These ligands interact with a number of different tyrosine kinase receptors (flt-1, flk-1, and flt-4) (13). VEGFs are expressed in cardiac myocytes and vascular smooth muscle cells, with increased expression in the setting of vascular injury, acute and chronic ischemia, and hypoxia (15). Their actions are mediated through downstream activation of Akt and eventual release of nitric oxide (NO) and include vascular permeability, increased endothelial cell growth and survival, and formation of tubular structures (13).

Preclinical data have provided evidence for VEGF as a proangiogenic agent in animal models of chronic myocardial ischemia with improvement in myocardial blood flow after VEGF treatment (16). Perivascular and intracoronary administration of VEGF has been demonstrated to improve myocardial flow and ventricular function in a porcine ameroid model of chronic ischemia (17). Because the actions of VEGF are mediated, in large part, through NO release, disease states that lead to diminished bioavailable NO and endothelial dysfunction, for example, hypercholesterolemia and diabetes, are associated with impairment in growth factor-induced angiogenesis (10).

Hypotension because of the release of NO and arteriolar vasodilation is associated with intravenous and intracoronary VEGF administration and has proven to be dose-limiting in phase I trials (18). A theoretical risk associated with growth factor administration is the development of plaque angiogenesis that may precipitate the growth and destabilization of atherosclerotic plaques (18). Based on the well-documented role of angiogenesis in tumor biology, accelerated growth of primary tumors and stimulation of metastasis is another theoretical concern (19). Proliferative retinopathy in the population of patients with diabetes is another disease with potential for pathological angiogenesis as a complication of growth factor therapy. These concerns provide support for local, rather than regional or systemic, delivery strategies. However, these matters have so far not become apparent clinically to date in phases I and II clinical trials (20).

Fibroblast growth factors

The FGF family consists of 23 proteins that are classified by their expression pattern, receptor binding preference, and protein sequence (21, 22). FGF is present in the normal myocardium (23). Its expression is stimulated by hypoxia (24) and hemodynamic stress (25). FGF-2 is a pluripotent molecule and modulates numerous cellular functions in multiple cell types. In the context of angiogenesis, it induces endothelial cell proliferation, survival, and differentiation and is also involved in the migration of endothelial cells, smooth muscle cells, macrophages, and fibroblasts (22). These effects are mediated through its interaction with the tyrosine kinase receptor FGFR-1, which also leads to the eventual downstream release of NO (26). However, in contrast to VEGF, a lesser number of studies have tied the angiogenic effects of FGF-2 directly to NO release. Additionally, FGF-2 stimulates endothelial cells to produce a variety of proteases, including plasminogen activator and matrix metalloproteinases (27, 28), promoting chemotaxis.

FGF was shown to induce angiogenesis in mature tissue by animal studies that demonstrated increased vascularity after intracoronary injections in acute coronary thrombosis models (29, 30). In studies using the ameroid constrictor model of chronic myocardial ischemia, FGF-2 therapy using perivascular and intrapericardial delivery improved coronary blood flow and regional left ventricular function (Fig. 2) (31, 32). Also, in studies using intracoronary infusions as the delivery method, improvements in myocardial perfusion and function were observed (33, 34). Similar to VEGF, FGF-2 can cause acute vasodilation and hypotension. In addition to the potentially adverse effects of growth factors mentioned above, a significant long-term side effect of high-dose FGF administration is renal insufficiency due to membranous nephropathy accompanied by proteinuria (20).

Fig. 2
Fig. 2:
The ameroid constrictor model of chronic myocardial ischemia is commonly used to evaluate the preclinical efficacy of growth factors and delivery strategies. This figure also demonstrates surgical implantation of FGF-2 sustained-release heparin-alginate capsules, which have been used in clinical trials of myocardial angiogenesis.

GROWTH FACTOR DELIVERY

Traditionally, two approaches have been used to achieve therapeutic angiogenesis: gene transfer and protein therapy. However, recently there has been increasing interest in cell-based therapy. These strategies will be briefly discussed.

Protein therapy

The advantages of protein therapy include controlled delivery, established safety, predictable pharmacokinetics and tissue levels, and absence of long-term unexpected side effects (35-37). The main disadvantages of this strategy include short tissue half-life of most proteins and high cost of recombinant molecules, shortcomings partly addressable with sustained delivery systems. An example of a sustained-release system is the use of heparin alginate capsules for perivascular FGF-2 delivery in surgical angiogenesis trials (4). Fig. 3 shows nuclear perfusion imaging in a patient treated with this method of FGF-2 delivery. It is important to note that some angiogenic agents cannot be delivered as proteins and thus may necessitate gene transfer. Examples of such agents are HIF-1α and PR39 (38, 39), which are transcription factors involved in the angiogenic cascade. However, for FGFs and VEGFs, protein therapy may supersede gene transfer, especially given the limitations of current vectors.

Fig. 3
Fig. 3:
Changes in myocardial perfusion as assessed by nuclear imaging before (top) and after (bottom) treatment with coronary artery bypass and perivascular FGF-2 therapy to the nonbypassed territory. This patient was treated with a left internal thoracic artery bypass to the left anterior descending artery, a saphenous vein bypass to an obtuse marginal branch of the circumflex coronary artery, and heparin alginate sustained-release capsules containing FGF-2 to the inferior wall territory (white arrows).

Gene therapy

The advantages of gene transfer include prolonged and sustained expression of the protein in the target tissues, ability to express transcriptional factors, potential for regulated expression, and ability to express multiple genes simultaneously. The major shortcomings of gene transfer are the short- and long-term toxicities and side effects of various vectors that are incompletely understood. Furthermore, gene-based therapy can potentially cause detrimental sustained expression, potentially leading to pathologic angiogenesis, inflammatory reaction to delivery vectors, and the potential for mutated pathogenic vectors (for viral therapy). Although the overall experience with protein-based (growth factor) therapy has been more extensive than gene transfer, several phases I and II studies involving gene therapy have recently been published (40-42).

Cell-based therapy

Cell-based gene transfer is a promising new strategy that uses autologous cells or cell lines transfected with a transgene of interest to express that transgene in vivo. The advantage of such a system is to circumvent the inflammatory response by using autologous cells and achieve prolonged expression by stable transfection using various measures, including electroporation, in vitro retroviral, or lentiviral transfection (43-48). In addition, complex constructs can be built that would allow stable regulated expression and multiple transgene expression. There are numerous investigations in progress studying the use of cellular transplantation for myocardial regeneration or angiogenesis using hematopoietic cells, skeletal myoblasts, and endothelial progenitor cells. In addition to inducing neovascularization in the target tissue, cell transplantation has a larger goal of tissue regeneration leading to functional improvements. As a result, clinical trials studying cell-based therapy focus primarily on improvements in myocardial function.

Cell-based therapy is currently in its nascent stages and is limited by a number of issues. First, the rate of cell survival in the days to weeks after transplantation remains low (49). Attempts to improve cell survival have included the coadministration of growth factors that stimulate proliferation and prevent cell death. Second, the methods currently available to track transplanted cells are quite limited. In animal models, fluorescent and radioactive probes have been used (50), but these methods have not yet demonstrated feasibility in clinical studies. The ability to reliably track transplanted cells can provide tremendous insight into their eventual fate and their contribution to the improvements in function that have been demonstrated in some clinical studies. Finally, a better understanding of the mechanisms by which these cells cause improvements in myocardial function would allow for the manipulation of these mechanisms to enhance the effects of cell therapy. Currently, it remains unclear whether these cells phenotypically and functionally differentiate into cardiomyocytes or endothelial cells whether they induce neovascularization through secretion of growth factors leading to improved perfusion of viable, but ischemic, tissue. Thus, although cell-based therapy holds promise for myocardial regeneration in general and myocardial angiogenesis in particular, many questions still remain to be answered before it can be consistently and reliably used in patients.

DELIVERY STRATEGIES

Delivery strategies can usually be classified as systemic (intravenous), local/regional infusion (intracoronary for myocardial angiogenesis and intra-arterial for limb angiogenesis), local periadventitial delivery (catheter-based or surgical implantation), and intramyocardial (catheter-based or surgical for myocardial therapy, intramuscular for peripheral vascular disease). Although a wealth of information exists regarding vector development and angiogenic potential of various cytokines, the delivery of these agents to the heart and peripheral vasculature has been arbitrarily chosen and tested for efficacy without optimizing the volume to be delivered, the rate of infusion or injection, the biocompatibility with materials used, and the optimal mode of delivery for each compound and vector. Different delivery methods have been used by different investigators for gene transfer to the heart and peripheral vasculature ranging from intracoronary (51) to epicardial (42) and endocardial (40) injections. Intravenous administration has not been advocated particularly with findings from several investigations that in following gene expression after intravenous administration of an adenoviral construct, the distribution was highest in the kidney followed by lung, liver, brain, and heart (52). Intracoronary administration of adenoviral vectors has been studied by several investigators (51, 53-55); however, most studies have been performed with intramuscular (limb) and intramyocardial delivery (heart) (56-58). Nonetheless, these delivery strategies have not been optimized for the vector and agent used, and basic parameters such as volume to be injected, needle depth, rate of injection, biocompatibility, and vehicle used are still under investigation.

CLINICAL STUDIES

More than 1200 patients have been treated with either protein- or gene-based growth factor therapy as part of phase I and II trials. Table 1 lists the placebo-controlled studies of growth factory therapy for myocardial angiogenesis published to date, including the agent and delivery methods used. All the studies have used isoforms of either VEGF or FGF delivered using a variety of methods. The following sections summarize the methods, results, and limitations of published studies.

PROTEIN THERAPY

Phase I trials

The first phase I clinical trial of coronary angiogenesis demonstrated the safety of intramyocardial injection of 0.01mg/kg of FGF-1 (59). A total of 40 patients undergoing CABG of the internal mammary artery to left anterior descending coronary artery were randomized to receive intramyocardial injections of either 0.01 mg FGF-1 or placebo. All the patients had further stenoses of the left anterior descending coronary artery distal to the anastomosis. Coronary angiography 12 weeks after treatment showed increased capillary refill in patients that received FGF-1 compared with control patients. Follow-up 3 years later confirmed the safety and efficacy of the FGF-1. Mortality was similar in the control and treatment groups. The capillary network seen at 12 weeks posttreatment persisted on angiography. Additionally, echocardiography suggested improved left ventricular ejection fraction (LVEF) (60).

A preliminary study of surgically delivered intramyocardial FGF-2 that demonstrated the safety of this technique was followed by a phase II randomized, double-blind, placebo-controlled trial in which 24 patients undergoing CABG with ungraftable areas of myocardium were randomized to 10 μg of FGF-2, 100 μg of FGF-2, or placebo (4, 36). Slow-release heparin-alginate microcapsules, which released FGF-2 over 3 to 4 weeks, were implanted into the ischemic and viable but ungraftable myocardial region. Follow-up averaged 16 months with clinical assessment and nuclear perfusion imaging. There were no reports of recurrent angina or repeat revascularizations for the 100-μg FGF-2 group versus three reports of recurrent angina and two repeat revascularizations in the control group. Significant reductions in nuclear defect size were observed in the 100-μg group. After a mean follow-up of 32 months, significant benefits were reported in both myocardial perfusion and angina-free period in patients treated with growth factor at either dose compared with those receiving placebo. Nuclear perfusion scans revealed a persistent reversible or a new fixed defect in four of five patients who received placebo versus only one of nine patients treated with FGF-2 (P= 0.03). In addition, among patients that received FGF-2, a trend toward improved LVEF was observed (61).

Phase I trials evaluating both intracoronary and intravenous administration of FGF-2 to examine the safety and efficacy of these less invasive methods of delivery have also been conducted (62). These were open-label dose-escalating studies. Fifty-two patients with CAD and inducible ischemia who were deemed suboptimal candidates for either percutaneous transluminal coronary angioplasty or CABG received intracoronary FGF-2 at doses ranging from 0.33 to 48 μg/kg. Hypotension was dose-limiting, with 36 μg/kg being the maximally tolerated dose. At 6 months of follow-up, patients reported improvement in quality-of-life assessments and reduced angina frequency and improved exertional capacity scores. Significant improvements were seen in exercise treadmill time (ETT), LVEF, target wall thickening, and myocardial perfusion as measured by magnetic resonance imaging. However, there was no correlation between the dose used and the efficacy parameters studied. The lack of a control group and the open-label design of the study preclude conclusions regarding the efficacy of the treatment. Another study evaluated intracoronary delivery of FGF-2 in a randomized, placebo-controlled, dose-escalation phase I trial (63). A total of 25 patients were randomized at a 2:1 ratio to a single intracoronary dose of FGF-2 or placebo. FGF-2 therapy was associated with hypotension in two patients and bradycardia in three patients. The FGF-2-treated group showed significantly increased epicardial coronary artery diameter compared with controls, but no improvement in treadmill exercise tolerance was observed.

Several phase I trials of the safety and tolerability of VEGF have also been conducted. Two trials investigated intravenous and intracoronary delivery of recombinant VEGF using dose-escalation regimens (63-65) and a follow-up period up to 60days. The results of these studies demonstrated that VEGF delivered by intracoronary and intravenous routes was well tolerated and suggested dose-dependent improvements in myocardial blood flow by nuclear perfusion studies. As a whole, these results provide evidence for the safety of protein-based angiogenic therapy with VEGF and FGF and suggest the efficacy of this therapeutic modality.

Phase II trials

Despite the promising results seen in both preclinical and phase I trials, randomized, double-blind, controlled phase II trials have shown modest, if any, benefit. The VEGF in ischemia for vascular angiogenesis (VIVA) trial was a multicenter, randomized, double-blind, placebo-controlled study of an intracoronary and intravenous regimen of recombinant VEGF (66, 67) A total of 178 patients were randomized to low-dose, high-dose, or placebo groups. VEGF was delivered by an intracoronary infusion followed by intravenous infusions at 3-day intervals. Treadmill exercise time was the primary end point. No significant improvements were reported in ETT or angina class in the treatment groups compared with controls at 60 days of follow-up. The VEGF infusions were well tolerated by patients in the treatment groups. Follow-up after 1 year revealed a trend in sustained improvement in angina class that was not statistically significant. No increased risk for cancer or myocardial infarction was apparent (66). Of note, all three groups in the VEGF in ischemia for vascular angiogenesis trial had significant improvements in ETT, angina class, and quality-of-life measures from baseline, which demonstrates the significance of the placebo effect in patients with end-stage CAD. Long-term follow-up of patients in clinical trials has shown persistence of the placebo effect for up to 30 months (68).

The FGF-2 initiating revascularization support trial (FIRST) was a multicenter, randomized, double-blind, placebo-controlled phase II study designed to examine the safety, pharmacokinetics, and efficacy of FGF-2 (69, 70). A total of 337 patients who were poor candidates for percutaneous or surgical revascularization were randomized to treatment with 0-, 0.3-, 3-, or 30-μg/kg doses of FGF-2 by intracoronary route. Exercise tolerance test time was the primary end point. The mean change in exercise tolerance test time was not significantly different after 90 or 180 days between treatment and control groups. However, a statistically significant benefit in ETT was shown in patients older than 63 years. Angina frequency was significantly reduced as determined by the Seattle Angina Questionnaire at 90 days, but the difference was no longer significant at 180 days because of continued improvement of the control group. No significant difference was observed in stress nuclear imaging.

Although the long-term follow-up of the randomized, double-blind, controlled trial of surgical intramyocardial delivery of FGF-2 showed persistent improvement in time freedom from angina and nuclear perfusion at 32 months in test patients versus control patients (61), the study population was small, and a larger trial is needed to confirm these results.

These disappointing results can be attributed to a variety of factors. Recombinant proteins have a relatively short plasma half-life. Animal studies have demonstrated that less than 1% of 125 I-FGF-2 administered using the intracoronary route is deposited in the myocardium at 1 h. Even less remains at 24h (37). Furthermore, the effect of growth factors is known to be diminished in the presence of endothelial dysfunction, a common finding in patients with coronary disease (9).

GENE THERAPY

Phase I trials

The first clinical trial included five patients with intractable angina and inoperable coronary disease that failed conventional medical management (71). Each patient received an intramyocardial injection of VEGF165 plasmid through a small left anterior thoracotomy. There were no complications related to the administration of the plasmid. All five patients reported decreased nitroglycerin use between 10 and 60 days after treatment. Single-photon emission computed tomography (SPECT)-sestamibi demonstrated improved blood flow, and angiography showed increased collateral flow to previously ischemic areas. LVEF remained unchanged. This was followed by a larger nonrandomized, uncontrolled, dose-escalating trial to assess the safety and bioactivity of intramyocardial delivery of the VEGF165 plasmid (56). Twenty patients received the VEGF165 plasmid and were observed for 180 days. There were no intraoperative complications, but one patient experienced cardiac arrest on the second postoperative day and died 4 months later secondary to aspiration pneumonia. Plasma VEGF levels were measured. The levels peaked at day 14 and returned to baseline by 90 days. Again, improvement was demonstrated on SPECT-sestamibi perfusion scans in 13 of the 17 patients studied at 60 days. Improvement in collateral filling was also demonstrated by angiography.

Rosengart and colleagues conducted a phase I clinical trial in 21 patients using direct intramyocardial injection of adenovirus encoding VEGF121 (55, 72). Fifteen patients received the therapy in conjunction with CABG, and six received it as sole therapy. Patients were followed for 6 months, and there were no complications secondary to vector administration. Trends toward improvement in angina classification and treadmill exercise testing were seen at 6 months. Analysis of the 99m Tc-sestamibi images for wall motion at stress in the region of vector administration showed an improvement at 30 days in most patients. Coronary angiograms showed increased collaterals.

A phase I, open-label, dose-escalation study of VEGF-2 naked DNA delivered via direct myocardial injection through a thoracotomy was conducted by Fortuin et al (42). Thirty patients with end-stage coronary disease were recruited and received 200, 800, or 2000 μg of naked plasmid DNA as sole therapy. There were no adverse effects, and almost all patients in this open-label study experienced a reduction in angina frequency, Canadian Cardiovascular Society (CCS) angina class, and nitroglycerin use. However, there was no correlation between dose and clinical benefit, and there was no angiographic evidence of angiogenesis.

Attempts to create a percutaneous method of gene delivery to the myocardium followed. The NOGA system is a catheter that electromechanically maps the myocardium to allow distinction between infracted and normal myocardium. The first study was a single blinded pilot study in which VEGF-2 plasmid DNA was delivered through intramyocardial injections in three patients and compared with three patients that received placebo injections (73). Reduction in angina frequency and nitroglycerin use was significantly greater in the patients that received the VEGF-2 plasmid versus the controls at 1-year follow-up. Significant decreases in the area of ischemia and improved perfusion scores were seen in test patients at 90 days after treatment.

Phase II trials

The early success of the phase I trials led to follow-up with larger phase II double-blind placebo-controlled trials. Losordo et al (74). randomized 19 patients in a 2:1 ratio to either receive VEGF-2 plasmid injection or placebo via the NOGA system. Follow-up was performed at 12 weeks. CCS angina classification significantly improved in patients with the VEGF-2 plasmid injections versus no improvement in control subjects. Nitroglycerin use was decreased in both groups and was not statistically significant. The mean duration of exercise increased significantly at 12 weeks of follow-up in phVEGF-2-transfected patients but was unchanged in patients randomized to placebo. Electromechanical mapping demonstrated a reduction in the area of ischemic myocardium in the patients that received VEGF-2 plasmid injections. Patients in the control group demonstrated no change in the area of ischemia.

The angiogenic gene therapy trial was a double-blinded, phase I/II trial using intracoronary infusion of increasing doses of adenovirus encoding for FGF-4 (75). Seventy-nine patients were randomized to receive either placebo or one of five doses of Ad5-FGF-4. At 12 weeks of follow-up, exercise tolerance was not significantly increased in treatment groups over placebo. However, a subgroup analysis of patients with initial ETTs of 10 min or less did show a significant improvement in treated patients versus controls. There were no differences in stress-induced wall motion scores by echocardiography between baseline and 4 or 12 weeks.

The Kuopio Angiogenesis Trial was a randomized double-blinded trial of intracoronary delivery of VEGF165 gene transfer at the time of percutaneous transluminal coronary angioplasty (41). A total of 109 patients were included in the study. Thirty-seven patients received VEGF adenovirus, 28 patients received VEGF plasmid liposome, and 38 control patients received Ringer's lactate solution. Although several patients had complications at the time of the procedure or soon after, none of these were attributed to the gene therapy. Follow-up time was 6 months. Gene transfer to coronary arteries was feasible and well tolerated. The overall clinical restenosis rate was 6%. In quantitative coronary angiography analysis, the minimal lumen diameter and percent of diameter stenosis did not significantly differ between the study groups. However, myocardial perfusion showed a significant improvement in the VEGF-Adv-treated patients after the 6-month follow-up. An inflammatory response was transiently present in the VEGF-Adv group, with transient fever and increases in serum CRP and l-lactate dehydrogenase levels. No increases were detected in the incidence of serious adverse events in any of the study groups.

The Euroinject One study (40) randomized 80 "no-option" (end-stage CAD) patients with stress-induced myocardial perfusion defects to intramyocardial plasmid gene transfer of VEGF165 (0.5 mg) or placebo, delivered percutaneously via the NOGA catheter system. This double-blinded trial demonstrated an improvement in wall motion abnormalities but no improvement in myocardial perfusion, assessed by 99m Tc sestamibi SPECT imaging. CCS angina class improved in both groups, with no significant difference between the two groups.

CHALLENGES IN MYOCARDIAL ANGIOGENESIS

Despite some disappointing initial results in clinical trials, therapeutic angiogenesis has the potential to provide new treatment strategies for patients with end-stage ischemic heart disease. Concerns regarding the safety of FGF and VEGF have not borne out in clinical trials. The trials described above have repeatedly demonstrated the safety of these agents as well as the safety of gene transfer. Angioma, neoplasms, plaque angiogenesis, and retinopathy have not been seen at the doses used in human trials. Challenges facing the future development of myocardial angiogenic therapy fall roughly into the categories described below.

Modifying the substrate

There are a variety of reasons why some of these trials failed to show improvement with treatment. Patients selected for these trials are possibly the ones most likely to fail to respond to therapeutic angiogenic agents. They have had multiple percutaneous and surgical revascularization attempts and have comorbidities such as diabetes mellitus, hypercholesterolemia, and endothelial dysfunction, which commonly accompany atherosclerotic disease. Because angiogenic growth factors act, in large part, through NO release, disease states that are associated with endothelial dysfunction because of decreased NO bioavailability can lead to an impaired angiogenic response. A diminished angiogenic response to ischemia and growth factor therapy has been demonstrated in the presence of endothelial dysfunction (Fig. 4) and may explain the disappointing results of clinical trials (9, 10, 76). Reversal of endothelial dysfunction by oral supplementation with l-arginine (an NO donor) in animal models has been shown to improve the angiogenic response (77-79). Thus, proendothelial agents can potentially be administered along with angiogenic growth factors to enhance the angiogenic response. A clinical trial evaluating the coadministration of l-arginine with surgical VEGF delivery is currently ongoing (80).

Fig. 4
Fig. 4:
Antiangiogenic influences observed in a porcine model of diet-induced hypercholesterolemia. Impaired coronary microvessel relaxation in the ischemic territory of hypercholesterolemic swine is evident in response to adenosine diphosphate (A), VEGF (B), and sodium nitroprusside (C), suggesting endothelial and smooth muscle dysfunction and decreased NO bioavailability. This is associated with reduced collateral-dependent myocardial perfusion (D) and increased myocardial expression of antiangiogenic protein endostatin (E).(76) *P < 0.05.

In addition to endothelial dysfunction, conditions such as hypercholesterolemia and diabetes are also associated with other antiangiogenic influences. In a preclinical swine model of chronic ischemia, hypercholesterolemia has been associated with increased expression of antiangiogenic protein, endostatin, and increased oxidative stress (76). The impaired angiogenic response in the presence of hyperglycemia has been associated with increased expression of another antiangiogenic protein, angiostatin (81), and increased glycation of the extracellular matrix (82). These antiangiogenic influences may become future targets for intervention with the goal of enhancing the effects of angiogenic therapy.

Optimal delivery of the ideal angiogenic agent

The phase II trials reported to date have primarily focused on intravascular delivery, with a few exceptions. Phase I trials of intramyocardial protein and gene delivery have borne promising results. Adequately powered, randomized, double-blind, placebo-controlled trials of intramyocardial delivery techniques are needed. Although intravenous and intracoronary delivery techniques are less invasive than surgical techniques, they result in systemic release and side effects such as NO-mediated hypotension, which limit the dose delivered. Intramyocardial delivery by a percutaneous, catheter-based, or a minimally invasive surgical procedure is appealing for the ability to target desired areas of the heart with the adequate dose and duration of treatment while avoiding systemic effects. Furthermore, it has become clear that the timing and duration of growth factor therapy may be critical in inducing an angiogenic response (83). Thus, prolonged exposure to growth factors may be required, which would necessitate the use of sustained release systems or gene transfer for growth factor delivery.

Finally, multiagent therapy may be needed to modulate the complex process of angiogenesis in humans. A synergistic mechanism of action between growth factors in angiogenesis has been suggested (84). Gene transfer methods using transcription factors such as HIF-1α that regulate the expression of multiple angiogenic genes may be an alternative strategy.

Clinical trial design and outcome assessment

Although hard end points, for example, myocardial infarction and death, provide objective measures of outcome, these events occur at a low frequency, and therefore, a prohibitively large study population may be required to show significant reductions in these end points. Changes in angina class, frequency, quality of life, and exercise tolerance are all somewhat subjective and therefore susceptible to the powerful placebo effect observed in these patients. Therefore, adequate blinding is critical to the ascertainment of a true therapeutic effect. The availability of a noninvasive, objective, and sensitive instrument to assess the efficacy of angiogenic therapy can help to address some of the above issues. SPECT, positron emission tomography, and magnetic resonance imaging are potential candidates for such an instrument.

As we further our knowledge of the basic mechanisms of angiogenesis and the techniques of angiogenic therapy, the strategies used in the design of technologies and clinical trials will be based on a more sound scientific foundation, which may allow therapeutic angiogenesis to become a reality in the treatment of CAD.

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Keywords:

Coronary artery disease; angiogenesis; endothelium; vascular endothelial growth factor; fibroblast growth factor

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