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Despite progressive insights into the pathologies underlying coronary, cerebral, and peripheral artery atherosclerosis, these conditions continue to cause critical tissue ischemia and disability on an epidemic scale. For the past several decades, research and therapeutic development have focused on preventing or reversing occlusive disease in conduit vessels. The ultimate failure of macrovessel-targeted therapies is never more evident than in peripheral arterial disease, in which progressive disease leads to amputation at rates that have not changed significantly in 30 years. Despite modern therapy, up to 8 million Americans with peripheral arterial disease are devastated by immobility, intractable ischemia, ulceration, impaired wound healing, or amputation,1 and the lack of additional treatment options leaves many patients with little hope for relief.
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The concept of therapeutic angiogenesis evolved from pioneering work in the 1970s by Folkman,2 who observed that the development and maintenance of an adequate microvascular supply is essential for the growth of neoplastic tissue. His hypothesis that the inhibition of “tumor angiogenic factors” would be effective against solid tumors was met with widespread skepticism, but 30 years of persistent research led to the development and approval of antiangiogenic treatments that now constitute a significant portion of the anticancer armamentarium. Soon after the identification of angiogenic growth factors, cardiovascular investigators began testing the hypothesis that stimulating angiogenesis could improve perfusion and function in ischemic tissues independent of macrovessel manipulation.3 Abundant preclinical data supported the safety and clinical potential of therapeutic angiogenesis that used growth factors or cellular-based strategies.4,5 Accordingly, given the grave prognosis and unrelenting disability associated with advanced peripheral arterial disease and the absence of predictive animal models, early-phase clinical studies commenced more than a decade ago. The evidence accumulated during phase 1 and phase 2 studies supports the safety of these approaches in humans6–21 (Table 1) and also provides indications of bioactivity in patients with these dreaded conditions6–27 (Table 2⇓⇓). Even so, true breakthroughs have been elusive. Why?
Table 1. Gene Therapy in Peripheral Arterial Disease: Safety Data
Table 2. Gene Therapy in Peripheral Arterial Disease: Bioactivity Data
Table 2. Continued
Table 2. Continued
In this issue of Circulation, Powell et al17 report on the safety and bioactivity profile of hepatocyte growth factor (HGF) plasmid injection for critical limb ischemia (CLI). In the double-blind, placebo-controlled, dose-escalating, multicenter HGF-STAT Trial, 104 patients with rest pain or tissue loss due to severe lower-extremity ischemia were assigned to receive injections of placebo or 1 of 3 dosing regimens of HGF plasmid into the ischemic leg muscle. A unique, prespecified analysis plan allowed the investigators to identify an increase in transcutaneous oxygen tension (TcPo2) in the high-dose group that was not present in other treatment groups, thus providing objective evidence for bioactivity. Other end points, such as amputation, wound healing, and ankle/brachial or toe/brachial index, did not reveal differences between treatment groups.
The results of this study and another recent report28 provide a degree of optimism for patients with CLI. They also illustrate the challenges inherent in implementing novel therapeutics in this patient population and can help guide the design of future studies. Patient enrollment is one major challenge in studies of gene therapy in patients with CLI, as evidenced by the fact that 2 of the largest randomized trials of angiogenic therapies to date required a total of 73 investigative sites and a cumulative span of ≈5 years to enroll 211 subjects. Thus, clinical trials are likely to be more expensive in patients with CLI than for many other conditions because of the need for large numbers of sites and the time required for enrollment of appropriately selected patients. A second key challenge for investigations in this patient population, which is nicely illustrated in the HGF-STAT study, is the fluctuating and somewhat unpredictable status of the CLI patient. CLI encompasses a range of disease severities and clinical trajectories, and even the most carefully selected patients will exhibit strikingly different clinical courses over short periods. Within a year of diagnosis, ≈20% of patients will die, 35% will require amputation, and the remainder will enter a more chronic state.29,30 Prospective definition of these subpopulations is problematic and often imprecise, and these difficulties must be addressed during trial design and data analysis. A similar challenge is encountered with variability within the more chronic group that tends to be represented in clinical trials. CLI challenges us to design studies in which a true signal of bioactivity will not be lost in the background noise of baseline variability.
Whether the method used in the present study, preselection of a subpopulation of patients for TcPo2 analysis, ultimately proves to be a useful surrogate that is predictive of clinical benefit remains to be seen, but documentation of bioactivity in a controlled trial is of paramount importance, and it is likely that the method applied by these investigators will be emulated and applied to other end points as well. In addition, the discordance between the surrogate and clinical end points observed in the study by Powell et al17 is prototypical of studies of angiogenic therapies. This is unsurprising when one considers the fact that all of the surrogate end points used in cardiovascular therapies have been validated for the detection and surveillance of large-vessel occlusive disease rather than for the accurate measurement of changes induced by angiogenesis. Thus, new methods and strategies may be required to accurately assess the benefits of therapeutic angiogenesis.
Safety analyses present yet another challenge for studies of patients with CLI. In the subgroup of 93 patients who were monitored for safety over 12 months in HGF-STAT, ≈50% experienced serious adverse events. This high frequency of serious adverse events underscores the severe clinical condition of CLI patients and the labor intensity and associated costs both of their care and of the management of clinical trials in this population. Clinical care alone of patients with CLI has been estimated at $43 000 per patient-year in 1990.31
Safety Aspects of Therapeutic Angiogenesis
Results from numerous randomized, controlled studies, including the study by Powell et al17 presented in this issue of Circulation, suggest that the transfer of proteins and genes to the human system is safe and feasible (Table 1). In the more than 1000 individuals who have been treated with gene therapy for therapeutic angiogenesis in phase I/II trials, adverse effects have generally been consistent with the baseline rate in the populations studied. However, until long-term safety data from large-scale investigations become available, these experimental therapies must be administered with scrupulous safety monitoring. A number of concerns need to be addressed, including the potential for angiogenesis-triggered malignancies, the impact of angiogenesis on physiological or pathological processes, and the specific adverse effects associated with each growth factor.
Because antiangiogenic therapies are effective for tumor treatment, it is logical to hypothesize that proangiogenic growth factors, in turn, might promote tumor development. Thus far, however, preclinical and clinical experiences with different growth factors have not identified an increased risk for malignancies, which appear to be related primarily to the older age of eligible patients. Stimulation of angiogenesis could also, in theory, induce or worsen retinopathy, as suggested by the high ocular fluid levels of vascular endothelial growth factor (VEGF) observed in patients with active proliferative diabetic retinopathy and by the successful treatment of this condition with anti-VEGF antibodies, but this problem has not materialized in clinical studies. There is concern that angiogenic factors may promote or destabilize atherosclerotic plaques by exerting angiogenic effects on the vasa vasorum; nevertheless, clinical studies have found no evidence of accelerated atherosclerosis in patients with advanced vascular/arteriosclerotic disease who were administered angiogenic cytokine therapy. Specific angiogenic factors have certain direct and predictable adverse effects. Some examples include hypotension after the administration of fibroblast growth factor-2 and VEGF proteins,32,33 which limited dosing in phase I trials; vascular leakage34 and transient tissue edema35 after VEGF and fibroblast growth factor gene transfer (although pedal edema usually responded well to diuretics36); and renal insufficiency with fibroblast growth factor-2 treatment, likely caused by membranous nephropathy. The adverse effect profile of HGF has not been characterized, but its ability to stimulate multiple downstream factors, which is the source of much enthusiasm for this agent, indicates that ongoing surveillance for safety is particularly important.
Aspects of Bioactivity in Therapeutic Angiogenesis
The elusiveness of a clinical breakthrough with angiogenic gene therapy may be explained by biological, technical, methodological, or disease-related factors (Table 2⇑⇑); for example, the poor efficacy obtained with intravascular administration of recombinant protein is likely caused by insufficient levels of growth factor in the targeted tissue. However, angiogenic gene therapy remains attractive because of the convergence of several features and perceptions. First, gene therapy offers a biological solution to a biological problem. Second, single genes appear to activate potent angiogenic mechanisms. Third, the angiogenic effect may be multiplied by the administration of morphogens that activate specific targets and pathways. Fourth, treatments can be designed to counteract specific pathological mechanisms. Fifth, delivery of the therapeutic agent can be restricted to the disease locus, thereby (presumably) maximizing the potential benefit while minimizing the occurrence and severity of side effects. Lastly, the function of specific organs can be safely enhanced with targeted, short-term transgene expression.
In retrospect, the success of gene therapy in animal models of vascular disease may have raised expectations to unreasonable heights. Because clinical efforts are extrapolated from preclinical studies, species-specific variations are unavoidable. Furthermore, chronic disease has progressed for decades in most patients and is often polygenetic, so successful treatment with a 1-time administration of a single gene appears unrealistic. The clinical success or failure of vascular gene therapy is also determined by the gene administered, the delivery vector and method of administration, and the underlying illness. Currently, preclinical studies are under way to identify the potential for complementary or synergistic effects from combinations in hopes of identifying the most efficient and safe biological “cocktail.” Viral vectors are generally effective for delivering genes, but immunogenic and pathogenic concerns have spurred efforts to find alternatives or novel virus serotypes. The transfection efficiency of nonviral vectors, on the other hand, is low and consequently presents a different set of challenges for in vivo applications that require a long-term effect. Novel formulations of DNA vectors may enable more efficient transfection, and methods that target nonviral vectors to specific tissues could increase efficiency and reduce adverse effects; however, targeted administration may be subject to technical limitations. For example, catheters are effective for local delivery to focal lesions in the vasculature or tissue of interest, but their usefulness depends on the target organ and its underlying pathology. Finally, a precise understanding of the mechanisms underlying neovascularization, including the time course and sequential roles of angiogenic and trophic factors, will enable researchers to better mimic the endogenous regenerative response.
Future Perspectives
Over the past 2 decades, angiogenic gene therapy has developed slowly but steadily as our mechanistic understanding of the factors involved has grown and as the selection of genes and vectors, the methods of administration, and clinical trial design continue to be refined. However, advancement from the safety-and-feasibility stage to routine clinical use will require carefully designed, adequately powered, large-scale, randomized, controlled phase II/III trials that incorporate end points that address methodological improvements, long-term safety, and bioactivity. Novel genetic targets will continue to be identified, and related factors capable of enhancing or attenuating the potency of gene therapy may be revealed. Investigators may also begin to assess the application of gene therapy in other areas of cardiovascular medicine, such as the prevention of postinterventional vascular remodeling and bypass-graft failure, stabilization of vulnerable plaques and aneurysms, or the treatment of hypertension, hyperlipidemia, and thrombotic states. Implicit in the above is the need to acknowledge the accumulated evidence of safety and to begin the pursuit of gene therapy applications at earlier stages of disease, when the potential for observable benefit may be enhanced.
Although the beneficial mechanisms of cell therapy are not completely understood, the potential of leveraging both the native expression of certain factors via gene therapy and augmenting an innate cellular response is attractive as a means to overcome methodological and technical challenges and yield synergistic effects, permitting lower doses of each therapeutic agent and the potential for enhanced safety.37 In theory, cell and gene therapy can be combined in 3 ways: (1) gene therapy supplemented with drug-induced stem cell or progenitor cell mobilization, (2) combined administration of both gene therapy and cell therapy, and (3) administration of genetically modified cells.
We believe that systematic efforts at the bench and the bedside will eventually lead to the routine clinical use of gene therapy for therapeutic angiogenesis. The development of gene therapy for other indications has progressed slowly; however, numerous targets for genetic modification can be envisioned.38 As new genetic targets are characterized and as our understanding of angiogenic mechanisms becomes more sophisticated, combinations of factors and/or the inclusion of other emerging strategies, such as cell therapy and bionanotechnology, may enhance patient response by inducing complementary or synergistic effects. For now, we can continue to learn from each clinical trial, and the work by Powell et al17 has provided important lessons that will inform the approach to future studies.
Acknowledgments
We thank W. Kevin Meisner, PhD, ELS, for editorial support and Mickey Neely for administrative support.
Sources of Funding
This work was supported in part by National Institutes of Health grants HL-53354, HL-57516, HL-77428, HL-63414, HL-80137, and PO1HL-66957. Dr Tongers was supported by the German Heart Foundation and Solvay Pharmaceuticals. Dr Roncalli was supported by the French Federation of Cardiology.
Disclosures
None.
Footnotes
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The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
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