Gene therapy for peripheral arterial diseases

Therapeutic angiogenesis using angiogenic growth factor is expected to be a new treatment for with patients with critical limb ischemia. The first human clinical trial treating peripheral vascular disease was started in 1994 using vascular endothelial growth factor (VEGF). To date, other potent angiogenic growth factors, such as fibroblast growth factor(FGF) or hepatocyte growth factor(HGF), have been also estimated in clinical trials for peripheral arterial disease. Several results from phase 1 or 2 trials using VEGF, FGF and HGF gene were encouraging. Phase 3 trials are now ongoing and their results are expected.     

The latest review of therapeutic angiogenesis using angiogenic growth factors to peripheral arterial diseases

Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment of patients with severe ischemic diseases. Indeed, human gene therapy for peripheral arterial disease(PAD) using VEGF gene demonstrated the beneficial effects. In contrast, we have reported the potent angiogenic activity of hepatocyte growth factor (HGF) in animal study and we planned gene therapy for ASO and Buerger disease using HGF gene (TREAT-HGF). In a prospective, open-labeled clinical trial, we investigated the safety and biological efficiency of this gene therapy in patients with peripheral arterial disease(PAD) who had failed conventional therapy.     

Beperminogene perplasmid for the treatment of critical limb ischemia

Therapeutic angiogenesis for the treatment of ischemic disease can be attained through the delivery of recombinant growth factor proteins, through gene transfer or cell transplantation. Gene transfer associated with adenovirus or naked plasmid DNAs has been extensively studied in clinical trials. An investigational product, beperminogene perplasmid, is the naked plasmid DNA encoding the cDNA of human HGF, which has potent angiogenic activity. In several clinical trials, beperminogene perplasmid showed favorable safety and efficacy profile in the treatment of critical limb ischemia. This article reviews the results of pre-clinical and clinical studies of beperminogene perplasmid in the treatment of critical limb ischemia caused by peripheral arterial disease and Buerger’s disease.     

Therapeutic angiogenesis for ischemic diseases

The clinical consequences of peripheral arterial disease include pain on walking, pain at rest and loss of tissue integrity in the distal ischemic limbs. Although development of beneficial drugs and intervention devices do contribute to the treatment of this disease, critical limb ischemia is estimated to develop in 500 to 1,000 individuals per million per year. As angiogenic growth factors can stimulate the development of collateral arteries, a concept called "therapeutic angiogenesis" is now evaluated in the clinical fields. Recent progress in molecular biology has led to the development of gene therapy as a new strategy to treat a variety of cardiovascular diseases using angiogenic growth factors such as vascular endothelial growth factor (VEGF). Therapeutic angiogenesis using angiogenic growth factors is expected to be a new treatment for patients with severe ischemic heart or peripheral arterial disease.     

Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat and rabbit hindlimb ischemia models: preclinical study for treatment of peripheral arterial disease

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells, and acts as a survival factor against endothelial cell death. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. We have previously reported that intra-arterial administration of recombinant HGF induced angiogenesis in a rabbit hindlimb ischemia model. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy, due to its disadvantages. Initially, we examined the transfection of 'naked' human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imaging, accompanied by the detection of human HGF protein. A significant increase in capillary density was found in rats transfected with human HGF as compared with control vector, in a dose-dependent manner (P < 0.01). Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid once on day 10 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (P < 0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-transfected animals. In addition, a significant increase in blood flow was assessed by a Doppler flow wire and the ratio in blood pressure of the ischemic limb to the normal limb was observed in rabbits transfected with HGF plasmid as compared with rabbits transfected with control vector (P < 0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hindlimb models, as potential therapy for peripheral arterial disease.     

Angiogenic therapy for coronary artery and peripheral arterial disease

Neovascularization in chronically ischemic adult cardiac and skeletal muscle results from the processes of angiogenesis, arteriogenesis and vasculogenesis. Therapeutic angiogenesis describes an emerging field of cardiovascular medicine whereby new blood vessels are induced to grow to supply oxygen and nutrients to cardiac or skeletal muscle rendered ischemic as a result of progressive atherosclerosis. Various techniques have been utilized to promote new blood vessel growth in the heart and extremities, including mechanical means such as surgical or percutaneous myocardial laser revascularization, angiogenic growth factor therapies involving members of the vascular endothelial growth factor and fibroblast growth factor families, and more recently, cellular-based therapies using stem cells known as endothelial progenitor cells or angioblasts. The following review discusses each of these treatment strategies in detail including both preclinical and clinical data for their use in peripheral arterial and coronary artery disease.     

Angiogenic therapy for coronary artery and peripheral arterial disease

Neovascularization in chronically ischemic adult cardiac and skeletal muscle results from the processes of angiogenesis, arteriogenesis and vasculogenesis. Therapeutic angiogenesis describes an emerging field of cardiovascular medicine whereby new blood vessels are induced to grow to supply oxygen and nutrients to cardiac or skeletal muscle rendered ischemic as a result of progressive atherosclerosis. Various techniques have been utilized to promote new blood vessel growth in the heart and extremities, including mechanical means such as surgical or percutaneous myocardial laser revascularization, angiogenic growth factor therapies involving members of the vascular endothelial growth factor and fibroblast growth factor families, and more recently, cellular-based therapies using stem cells known as endothelial progenitor cells or angioblasts. The following review discusses each of these treatment strategies in detail including both preclinical and clinical data for their use in peripheral arterial and coronary artery disease.     

Gene therapy for therapeutic angiogenesis in critically ischaemic lower limb - on the way to the clinic

Currently, no effective pharmacological treatment is available for vascularisation defects in lower limbs. Many patients presenting with persistent pain and ischaemic ulcers are not suitable candidates for surgical or endovascular approaches. Further refinement of the available methods will undoubtedly lead to a more active approach towards treatment of peripheral arterial occlusive disease (PAOD). Recently, therapeutic angiogenesis, in the form of recombinant growth factor administration or gene therapy, has emerged as a novel tool to treat these patients. However, improved gene transfer methods and better understanding of blood vessel formation are required to bring therapeutic angiogenesis to clinical practice. Here we review the clinical problem (PAOD), mechanisms of blood vessel formation (angiogenesis, vasculogenesis and arteriogenesis), experimental evidence and clinical trials for therapeutic angiogenesis in critically ischaemic lower limbs. Also, angiogenic growth factors, including vascular endothelial growth factors (VEGFs) and fibroblast growth factors (FGFs), delivery methods, and vectors for gene transfer in skeletal muscle, are discussed. In addition to vascular growth, gene transfer of growth factors may enhance regeneration, survival, and innervation of ischaemic skeletal muscle. Nitric oxide (NO) appears to be a key mediator in vascular homeostasis and growth, and a reduction in its production by age, hypercholesterolemia or diabetes leads to the impairment of ischaemic disorders.     

Pro- and anti-angiogenic therapy and atherosclerosis with special emphasis on vascular endothelial growth factors

INTRODUCTION: Atherosclerosis is a complex, progressive disease affecting nearly half of the population in Western countries. Although several treatment methods are already available, they may not be applicable to all patients suffering from advanced cardiovascular diseases, such as end-stage myocardial ischemia or difficult peripheral ischemia and potential new treatment methods are under intensive investigation. AREAS COVERED: VEGFs are major angiogenic molecules controlling vascular growth and function, vascular homeostasis, permeability and vasodilatation. Therefore, they have been regarded as potential new treatment agents for ischemic heart and peripheral vascular disease and several pro-angiogenic clinical trials have been conducted. In contrast, VEGFs also take part in pathological states by inducing microvessel growth in, for example tumors and atherosclerotic lesions. In this review, the biological basis of atherosclerosis and VEGF biology are presented as well as the latest results from pre-clinical research and clinical trials of pro- and anti-angiogenic therapy. EXPERT OPINION: Even though pro-angiogenesis has been shown to be safe and well-tolerated in clinical trials, efficacy of the treatment has not been satisfactory. In the expert opinion section of the review, we discuss the major obstacles to cardiovascular gene therapy and some future prospects.     

Gene-therapy for peripheral vascular diseases

The prognosis for patients with chronic critical leg ischemic is often poor. The treatment of peripheral vascular disease, although greatly improved over recent decades by drug medication, surgical and minimally-invasive techniques, remains limited by vascular proliferative lesions and by our inability to modulate the progression of native disease. The therapeutic angiogenesis is now the most expected therapy for peripheral vascular diseases. This review explores some of concepts and methods of therapeutic angiogenesis including gene therapy using angiogenic growth factor such as VEGF and basic FGF and an implantation of bone marrow derived endothelial progenitor cells.     

Hepatocyte growth factor as potential cardiovascular therapy

Hepatocyte growth factor is a mesenchyme-derived pleiotropic factor that regulates the growth, motility and morphogenesis of various types of cells, and is also a member of the angiogenic growth factors. Hepatocyte growth factor is secreted by vascular endothelial cells and smooth muscle cells, and the hepatocyte growth factor receptor, c-met, was also observed in these vascular cells. Treatment of human aortic endothelial cells with recombinant hepatocyte growth factor resulted in a significant increase in cell proliferation, accompanied by mitogen-activated protein kinase and Akt/protein kinase B phosphorylation. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As preclinical study of gene therapy using hepatocyte growth factor to treat peripheral arterial disease, naked hepatocyte growth factor plasmid was intramuscularly injected into the ischemic hind limb of rabbits in order to evaluate its angiogenic activity. Intramuscular injection of hepatocyte growth factor plasmid once on day 10 following surgery, produced significant augmentation of collateral vessel development in the ischemic limb on day 30. In the clinical setting, the authors further investigated the safety and efficacy of hepatocyte growth factor plasmid DNA in patients with critical limb ischemia, in a prospective open-labeled trial. Intramuscular injection of naked plasmid DNA was performed in the ischemic limbs of six patients with critical limb ischemia with arteriosclerosis obliterans (n = 3) or Buerger disease (n = 3) graded as Fontaine III or IV. In the efficacy evaluation, a reduction of pain scale of more than 1 cm on a visual analog pain scale was observed in five out of six patients. An increase in ankle pressure index of more than 0.1 was observed in five out of five patients. The long diameter of eight out of 11 ischemic ulcers in four patients was reduced by more than 25%. Intramuscular injection of naked hepatocyte growth factor plasmid is safe, feasible and can achieve successful improvement of ischemic limbs. Although the present data were obtained to demonstrate safety in a Phase I/early Phase II trial, the initial clinical outcome with hepatocyte growth factor gene transfer seems to indicate its usefulness as sole therapy for critical limb ischemia. Randomized placebo-controlled clinical trials of alternative dosing regimens of gene therapy will be required to define the efficiency of this therapy.     

Hepatocyte growth factor as potential cardiovascular therapy

Hepatocyte growth factor is a mesenchyme-derived pleiotropic factor that regulates the growth, motility and morphogenesis of various types of cells, and is also a member of the angiogenic growth factors. Hepatocyte growth factor is secreted by vascular endothelial cells and smooth muscle cells, and the hepatocyte growth factor receptor, c-met, was also observed in these vascular cells. Treatment of human aortic endothelial cells with recombinant hepatocyte growth factor resulted in a significant increase in cell proliferation, accompanied by mitogen-activated protein kinase and Akt/protein kinase B phosphorylation. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As preclinical study of gene therapy using hepatocyte growth factor to treat peripheral arterial disease, naked hepatocyte growth factor plasmid was intramuscularly injected into the ischemic hind limb of rabbits in order to evaluate its angiogenic activity. Intramuscular injection of hepatocyte growth factor plasmid once on day 10 following surgery, produced significant augmentation of collateral vessel development in the ischemic limb on day 30. In the clinical setting, the authors further investigated the safety and efficacy of hepatocyte growth factor plasmid DNA in patients with critical limb ischemia, in a prospective open-labeled trial. Intramuscular injection of naked plasmid DNA was performed in the ischemic limbs of six patients with critical limb ischemia with arteriosclerosis obliterans (n = 3) or Buerger disease (n = 3) graded as Fontaine III or IV. In the efficacy evaluation, a reduction of pain scale of more than 1 cm on a visual analog pain scale was observed in five out of six patients. An increase in ankle pressure index of more than 0.1 was observed in five out of five patients. The long diameter of eight out of 11 ischemic ulcers in four patients was reduced by more than 25%. Intramuscular injection of naked hepatocyte growth factor plasmid is safe, feasible and can achieve successful improvement of ischemic limbs. Although the present data were obtained to demonstrate safety in a Phase I/early Phase II trial, the initial clinical outcome with hepatocyte growth factor gene transfer seems to indicate its usefulness as sole therapy for critical limb ischemia. Randomized placebo-controlled clinical trials of alternative dosing regimens of gene therapy will be required to define the efficiency of this therapy.     

Angiogenic and antifibrotic actions of hepatocyte growth factor improve cardiac dysfunction in porcine ischemic cardiomyopathy

Impairment of cardiac function in ischemic cardiomyopathy has been postulated to be due to the decrease in blood flow and increase in collagen synthesis. Therefore, an approach to alter them directly by means of a growth factor may open up a new therapeutic concept in ischemic cardiomyopathy. From this viewpoint, hepatocyte growth factor (HGF) is a unique growth factor with angiogenic and antifibrotic effects. Thus, we examined the feasibility of gene therapy using HGF plasmid DNA for ischemic cardiomyopathy. Human HGF plasmid DNA at a dose of 0.4 or 4 mg was injected into ischemic myocardium of pigs induced by ameroid constrictor with the NOGA system. At 1 month after injection, the ischemic area was significantly reduced in the HGF group, accompanied by a significant increase in capillary density and regional myocardial perfusion in the ischemic area (P<0.01). In contrast, a significant decrease in fibrotic area was observed in the HGF group, associated with a significant decrease in collagen I, III and TGF-beta synthesis as compared to the control group (P<0.01). Consistently, cardiac function was significantly improved in the 4 mg HGF group as compared to the control group (P<0.05). Overall, the present in vivo experiments demonstrated that intramyocardial injection of human HGF plasmid DNA in ischemic cardiomyopathy resulted in a significant improvement in cardiac function through an increase in blood flow and decrease in fibrosis. These favorable outcomes suggest potential utility to treat patients with ischemic heart disease using HGF gene transfer. Currently, a phase I study using human HGF plasmid DNA is ongoing to test the validity of this concept.     

Angiogenic gene therapy strategies for the treatment of cardiovascular disease

Coronary artery disease (CAD) and peripheral vascular disease (PVD) are significant medical problems worldwide, and arguably the biggest medical problems in the developed world. Although substantial progress has been made in prevention as well as in the treatment of these diseases, particularly of CAD, there are a large number of patients, who despite maximal medical treatment, have substantial symptomatology, and who are not candidates for mechanical revascularization. Therapeutic angiogenesis represents a novel, conceptually appealing, treatment option for these patients. Consequently, there are several different products in clinical trials, looking at various angiogenic growth factors. A number of small, mostly open-labeled phase I or phase I/II studies have been conducted with adeno- and plasmid-based vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) gene constructs in CAD and PVD. Although these studies have provided intriguing indications that new vessel formation is possible, and that these new vessels could be functional, these studies have been too small to allow conclusions to be drawn about potential efficacy. A number of proof-of-concept studies are presently underway or planned with four different constructs Ad(GV)VEGF121.10 (BioByPass; GenVec Inc), ph-VEGF (St Elizabeth's Medical Center of Boston Inc), Ad5-FGF4 (Collateral Therapeutics Inc/Schering Inc) and NV1FGF (Aventis Pharma AG/Aventis Gencell), and should, upon completion, provide a better indication as to the potential therapeutic role of these treatment modalities in the armamentarium against atherosclerotic disease. This exciting new field is reviewed, with special emphasis on clinical trials.     

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.     

Gene therapy for peripheral arterial disease

Introduction: Gene therapy has emerged as a novel therapy to promote angiogenesis in patients with critical limb ischemia (CLI) caused by peripheral artery disease. Researchers working in this area have focused on pro-angiogenic factors, such as VEGF, fibroblast growth factor (FGF) and hepatocyte growth factor (HGF). Based on the elaborate studies and favorable results of basic research using naked plasmid DNA (pDNA) encoding these growth factors, some clinical Phase I and Phase II trials have been performed. The results of these studies demonstrate the safety of these approaches and their potential for symptomatic improvement in CLI patients. However, the Phase III clinical trials have so far been limited to HGF gene therapy. Because one pitfall of the Phase III trials has been the limited transgene expression achieved using naked pDNA alone, the development of more efficient gene transfer systems, such as ultrasound microbubbles and the needleless injector, as well as the addition of other genes will make these novel therapies more effective and ease the symptoms of CLI.

Areas covered: This study reviews the previously published basic research and clinical trials that have studied VEGF, FGF and HGF gene therapies for the treatment of CLI. Adjunctive therapies, such as the addition of prostacyclin synthase genes and the development of more efficient gene transfer techniques for pDNA, are also reviewed.

Expert opinion: To date, clinical studies have demonstrated the safety of gene therapy in limb ischemia but the effectiveness of this treatment has not been determined. Larger clinical studies, as well as the development of more effective gene therapy, are needed to achieve and confirm beneficial effects.