authors: 1Hong H. Keo, MD, 2Alan T. Hirsch, MD, 1Iris Baumgartner, MD, 3Sigrid Nikol, MD, 2Timothy D. Henry, MD ________________________________ Abstract Critical limb ischemia (CLI) represents the most severe stage of atherosclerotic lower extremity peripheral artery disease (PAD), and CLI prevalence is expected to increase as the population ages. The current standard of care for CLI relies on direct revascularization, either by endovascular techniques or open surgical approaches, as there are few effective medical treatments for this condition. Therapeutic angiogenesis is a novel approach to improving limb outcomes for these patients. Experimental preclinical studies and phase I/II clinical trials of therapeutic angiogenesis using gene transfer in patients with CLI unsuitable for revascularization have shown promising results. In this review, we describe the potential clinical impact of this new approach as an adjunct in our therapeutic armamentarium. Introduction Critical limb ischemia (CLI) represents the most advanced stage of atherosclerotic, lower extremity peripheral artery disease (PAD) and is associated with high rates of cardiovascular morbidity, mortality, and major amputation.1 The incidence of CLI is estimated to be 125,000 to 250,000 patients per year in the United States and is expected to grow as the population ages.2–5 The 1-year mortality rate of patients with CLI is 25% and may be as high as 45% in those who have undergone amputation.1,6 The current standard of care for individuals with CLI includes lower extremity revascularization, either through open peripheral surgical procedures, endovascular techniques, or lower extremity amputation (i.e., if revascularization has failed or is unfeasible).1,7 Despite advanced techniques in endovascular and surgical procedures, a considerable proportion of patients with CLI are not suitable for revascularization. Of these patients, 30% will require major amputation and 23% will die within 3 months.8 Therapeutic angiogenesis is a novel strategy under investigation for the treatment of PAD that utilizes angiogenic growth factors, genes to encode these growth factors, or stem cells to promote neovascularization, in an attempt to increase perfusion to ischemic tissues through various mechanisms of action.9 Early clinical trials of gene transfer for therapeutic angiogenesis have been promising and provide hope for CLI patients who are unsuitable candidates for revascularization. This paper will review the impact of therapeutic angiogenesis for the treatment of patients with CLI, including the safety and efficacy data provided by those clinical trials completed to date, and will outline potential future directions for clinical research. The Concepts Underlying Therapeutic Angiogenesis The concept of therapeutic angiogenesis evolved from pioneering work by Folkman,10 who observed that the development and maintenance of an adequate microvascular supply is essential for the growth of neoplastic tissue. Following the early 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.11 Therapeutic angiogenesis involves the administration of angiogenic growth factors, as recombinant protein or gene encoding for those growth factors or stem cells to augment the collateral circulation and enhance blood flow to ischemic tissues. Angiogenic protein growth factors have been utilized, but require intra-arterial delivery and have short half lives.12,13 In particular, for PAD, gene therapy has theoretical advantages of intramuscular delivery, permitting repeated administration and a more prolonged therapeutic effect. Angiogenic growth factors can be administered using non-viral or viral-vector encoding genes. The non-viral method uses naked plasmid DNA to transfer the gene encoding the desired angiogenic protein to the ischemic tissue. The viral delivery method uses viruses as a vector to introduce the new gene to the ischemic tissues, also referred to as transfection. The viruses most often used in angiogenic studies are adenoviruses.14 The major advantage of this method is that transfection of growth factors can be achieved with high efficiency. However, the potential downside of this method is that transfection efficiency may be limited by prior viral exposure, and repeated administration might not be efficacious, due to rapid degradation of the viral vector.15,16 Therapeutic angiogenesis and angiogenic growth factors. Therapeutic angiogenesis was first evaluated by Dr. Jeffrey Isner in a 71-year-old patient with severe PAD and great toe gangrene in 1994.17 Human plasmid phVEGF165 in a dose of 2 mg was applied to the hydrogel polymer coating of an angioplasty balloon. By inflating the balloon in the vascular lumen, plasmid DNA was transferred to the distal popliteal artery.17 Functional and angiographic parameters improved within 12 weeks, and spider angiomata and edema developed unilaterally in the affected limb, suggesting the treatment had a local angiogenic effect. Since this pioneering, experimental therapy numerous angiogenic growth factors have been developed and tested in clinical trials with demonstration of angiogenic potential.18 Table 1 provides an overview of angiogenic growth factors that have been identified to stimulate neovascularization in preclinical and clinical models. Of these, the following four have been thus far evaluated in clinical trials of patients with CLI. The most potent angiogenic factor affecting endothelial cell proliferation is vascular endothelial growth factor (VEGF), also known as vascular permeability factor. This factor has a high affinity for binding to endothelial cells. It was first discovered in the early 1980s as an agent that triggers hyperpermeability and macromolecule extravasation from microvessels.19 Four different receptors are known to bind the members of the VEGF family: VEGFR-1, VEGFR-2, VEGFR-3, and neuropilin-1.20 Endothelial cell specificity has been considered to represent an important advantage of VEGF for therapeutic angiogenesis because endothelial cells represent the critical cellular element responsible for new collateral vessel formation.21 Fibroblast growth factor (FGF) consists of 23 structurally related members and is important for the growth and migration of many cell types in the vessel wall, including endothelial cells and smooth muscle cells.22, 23 FGF is a key regulator of vessel growth and has successfully been used to promote angiogenesis.24 Hypoxia-inducible factor 1 (HIF-1) functions as a master regulator of oxygen and undergoes conformation changes in response to oxygen concentration. It is a heterodimeric transcription factor complex that consists of two subunits, HIF-1a and HIF-1.25,26 HIF-1aβ is expressed in the cell nucleus and its activity is not controlled by oxygen homeostasis, whereas the activity of HIF-1 is regulated by oxygen levels.27 Human endothelial cells transfected with Ad2/HIF-1a/VP16 (Ad, adenovirus; VP16, herpes simplex virus VP transaction domain) have been shown to promote endothelial proliferation and tube formation as a result of up-regulation of the expression of multiple angiogenic factors.28 Hepatocyte growth factor (HGF) is a mesenchymal-derived pleiotropic factor that regulates cell growth, cell motility, and morphogenesis of various types of cells. HGF is also a powerful angiogenic growth factor and is secreted by vascular endothelial cells and smooth muscle cells.29 Angiogenic Activity and Clinical Efficacy Vascular endothelial growth factors. After introducing feasibility of angiogenic gene transfer of VEGF165 using human plasmid as a vector in 1994,17 Isner and coworkers30 conducted a non-randomized phase I clinical trial to document the safety of intramuscular phVEGF165 gene transfer in 6 patients (7 limbs) with CLI due to Buerger’s disease and who were not candidates for catheter or surgical revascularization. VEGF165 plasmid DNA was administered into the calf or distal thigh muscles with doses of 2 x 2 mg. After 14-month follow up, evidence of improved perfusion to the distal ischemic limb was documented by an increase in ankle-brachial index (ABI) in 3 limbs and by magnetic resonance imaging (MRI) in all 7 limbs, as well as by newly visible collateral vessels formation shown on serial contrast angiography in 7 of the 7 limbs.30 In another phase I study, 9 patients (10 limbs) received intramuscular injection of phVEGF165 into the ischemic muscle in a dose of 2 mg and repeated similar injection was performed after 4 weeks of initial injection.21 After 6-month follow up, evidence of improved perfusion was documented by increased ABI values, contrast angiography revealing newly visible collateral blood vessel formation, improved distal flow on magnetic resonance angiography, and ulcer healing and limb salvage in 3 patients who would have been amputated otherwise.21 Shyu et al31 conducted a phase I clinical study of 21 patients (24 limbs) with CLI unsuitable for catheter or surgical revascularization using plasmid DNA to deliver VEGF165 into the ischemic muscle in a dose escalation of 0.4, 0.8, 1.2, 1.6, and 2.0 mg at baseline and at 4 weeks thereafter. After 6-month follow up, evidence of improved perfusion was documented by ABI values (0.58 ± 0.24 to 0.72 ± 0.28; p Safety Aspects of Gene Therapy The inclusion criteria for these trials have generally included Rutherford class 4 and 5 (rest pain or nonhealing ulcers) with documented abnormal ankle or toe pressure or TcPO2 who were not amenable (or suboptimal) for standard surgical or percutaneous revascularization. In general, patients were excluded if they had undergone a successful revascularization procedure within the past 3 months, or had a history of cancer within the last 5 years, severe retinopathy, or macular degeneration. In addition, patients are required to have routine cancer screening, as recommended by the American Cancer Society, and ophthalmologic evaluations. Other more standard exclusion criteria include chronic hemodialysis, immunosuppressive medication, chronic inflammatory disease, bleeding disorders, imminent major amputation, women of child-bearing potential, hepatic disease, HIV-positivity, or life expectancy of less than 1 year.9,37,39,41 Therefore, these have been high-risk CLI patients who have limited treatment options, but who have been screened to eliminate potential adverse effects. Results from numerous randomized, controlled trials suggest that gene transfer is feasible and safe with no serious adverse effects apparent at this time. Adverse effects have generally been consistent with the baseline rate for the population studied.18 However, long-term safety data from large scale trials is lacking. Potential adverse effects include angiogenic stimulation of unrecognized malignancies, progression of diabetic retinopathy, macular degeneration, or progression of atherosclerosis due to angiogenic effects on the vasa vasorum. At present, however, experimental and clinical experiences with different growth factors have not identified an increased risk for malignancies, retinopathy, or acute coronary syndromes.18 Other less SAE have been reported, which included hypotension, vascular leakage, and transient lower limb edema when FGF or VEGF is used.18,43 A transient increase in C-reactive protein, proteinuria, and thrombocytopenia and renal insufficiency has also been reported in other angiogenic trials.21,33,44,45 HGF in angiogenic trials showed no greater adverse effects than the baseline rate of the population studied, which makes this agent very interesting. Overall, the initial trials demonstrated an excellent safety profile. However, ongoing systematic surveillance for safety is clearly needed in this expanding research field. Future Perspectives Gene therapy has slowly continued to develop as a potential treatment over the past two decades and has provided important knowledge of different growth factors used, selection of genes and vectors and the methods of administration. Numerous angiogenic phase I and II trials have been published showing minimal toxicity and substantial clinical improvement demonstrated in patients with CLI. Large-scale phase III trials, such as the multicenter double-blind, placebo-controlled trial evaluating efficacy and safety of NV1FGF in CLI patients with skin lesions (TAMARIS),46 are underway, which will include 490 patients assigned to placebo or intramuscular injection of NV1FGF. This study is sponsored by Sanofi aventis (Sanofi-aventis, Paris, France) and is expected to be completed in July 2010. Another promising approach of therapeutic angiogenesis in patients with CLI unsuitable for revascularization is the use of cell-based therapy. There have been a number of early phase I clinical trials and several larger phase II randomized placebo-controlled trials are underway.47 Overall, despite the promising results of therapeutic angiogenesis in initial clinical trials, a large number of questions remain unanswered, including the optimal dose, dosing schedule, and route of administration. Given the complexity of angiogenesis, it is unclear which growth factor will have the most effective neovascularization potential and whether a combination of growth factors might be more effective. Although these challenges remain, we believe that the ongoing trials will enhance our understanding of angiogenic mechanisms and might yet lead to the routine clinical use of therapeutic angiogenesis. Our understanding of angiogenic mechanisms will undoubtedly become more sophisticated, and new treatment strategies may use combinations of growth factors and/or cell therapy using endothelial progenitor cell. This approach may enhance angiogenesis in a complementary or synergistic manner. Conclusion Both plasmid DNA and adenoviral vectors have successfully been used to deliver a variety of angiogenic growth factors in patients with CLI to promote neovascularization. Clinical phase I/II studies have shown excellent safety and promising efficacy in surrogate endpoints, such as pain, ulcer healing, and perfusion imaging. Although many questions remain unanswered, therapeutic angiogenesis has the potential to revolutionize our approach to patients with CLI. Acknowldegement. Dr. Keo has received a grant from Swiss National Science Foundation (PBBRB-121067). ___________________________ From 1University of Bern, Bern, Switzerland, 2Minneapolis Heart Institute Foundation at Abbott Northwestern Hospital, University of Minnesota School of Public Health, Minneapolis, Minnesota, 3Asklepios Klinik St, Georg, Germany. Address for correspondence: Timothy D. Henry, MD, Minneapolis Heart Institute Foundation Clinical Research-Suite 100, 920 E, 28th Street, Minneapolis, MN 55407. e-mail: firstname.lastname@example.org. Manuscript received March 13, 2009, provisional acceptance given May 26, 2009, accepted June 2, 2009. Dr. Henry has received speaker honoraria from Sanofi Aventis and research grants from ViaMed.