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Therapeutic Angiogenesis in Peripheral Arterial Disease: Can Biotechnology Produce an Effective Collateral Circulation?

  • D.J Collinson
    Affiliations
    Centre for Integrated Systems Biology and Medicine, School of Medical and Surgical Sciences University of Nottingham, Nottingham, UK
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  • R Donnelly
    Correspondence
    Corresponding author. Prof. Richard Donnelly, University of Nottingham Medical School, Derby City General Hospital, Derby DE22 3DT, UK
    Affiliations
    Centre for Integrated Systems Biology and Medicine, School of Medical and Surgical Sciences University of Nottingham, Nottingham, UK
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      Abstract

      The physiological processes of angiogenesis, vasculogenesis and arteriogenesis contribute to the growth of collateral vessels in response to obstructive arterial disease causing lower limb or myocardial ischaemia, but in clinical practice the endogenous angiogenic response is often suboptimal or impaired, e.g. by factors such as ageing, diabetes or drug therapies. Therapeutic angiogenesis is an application of biotechnology to stimulate new vessel formation via local administration of pro-angiogenic growth factors in the form of recombinant protein or gene therapy, or by implantation of endothelial progenitor cells that will synthesize multiple angiogenic cytokines. Numerous experimental and clinical studies have sought to establish ‘proof of concept’ for therapeutic angiogenesis in PAD and myocardial ischaemia using different treatment modalities, but the results have been inconsistent. This review summarises the mechanisms of angiogenesis and the results of recent trials evaluating the efficacy and safety of different gene therapy, recombinant protein and cellular-based treatment approaches to enhance collateral vessel formation.

      Keywords

      1. Introduction

      The strengths and limitations of surgical revascularization in peripheral arterial disease (PAD) are well recognised. In general, by-pass grafting and percutaneous interventions are reserved for patients with critical limb-threatening ischaemia and those with disabling claudication due to discrete, proximal disease.
      • Whitehill T.A
      Role of revascularization in the treatment of claudication.
      Vascular Surgical Society of Great Britain and Ireland
      Critical limb ischaemia: management and outcome. Report of a national survey.
      This leaves a significant number of patients with moderate-to-severe PAD with few, if any, effective treatment options to improve symptoms, restore distal perfusion and preserve tissue viability. This particularly applies to patients with diffuse and distal disease, who are often diabetic, and those who, despite successful revascularization, present with recurrent symptoms that are not amenable to further surgical intervention.
      There is good evidence that the development of collateral vessels has a favourable effect on the symptoms and outcomes of atherosclerotic disease. For example, in acute myocardial infarction the presence of a collateral circulation decreases infarct size and improves left ventricular function and patient survival.
      • Charney R
      • Cohen M
      The role of the coronary collateral circulation in limiting myocardial ischaemia and infarct size.
      Over the last 10 years, there has been considerable interest in the physiological mechanisms that regulate new vessel formation.
      • Risau W
      Mechanisms of angiogenesis.
      The main factors that stimulate growth of collateral vessels are, firstly, the duration and severity of ischaemia, as well as shear stress and inflammation, but numerous local and systemic cytokines also have pro- or anti-angiogenic effects. Cancer researchers have been particularly interested in blocking angiogenesis as a method of inhibiting tumour growth; meanwhile, cardiovascular researchers have explored the feasibility of enhancing collateral vessel formation in patients with chronic ischaemia of the myocardium or lower limb. This review describes the underlying mechanisms and principles of therapeutic angiogenesis, and summarises important results from recent clinical and experimental studies in PAD.

      2. Terminology Relating to New Vessel Formation

      Angiogenesis is the sprouting of new capillaries from existing vascular structures, a process that is triggered by endothelial cell migration and proliferation. Remodelling of the extracellular matrix (ECM), tubule formation and expansion of the surrounding vascular tissues are key elements of angiogenesis (Fig. 1) . Vasculogenesis, however, is quite different: the in situ formation of new blood vessels from circulating bone marrow-derived endothelial progenitor cells (EPCs) which differentiate into endothelial cells and fuse into luminal structures. It was previously assumed that vasculogenesis only occurred during embryological development, but there is increasing evidence that neovascularization in adult tissues involves both processes of angiogenesis and vasculogenesis.
      • Asahara T
      • Masuda H
      • Takahashi T
      • Kalka C
      • Pastore C
      • Silver M
      • et al.
      Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological neovascularization.
      • Isner J.M
      • Asahara T
      Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization.
      Under normal conditions, the number of circulating EPCs is relatively small but vascular trauma or ischaemia results in mobilization and proliferation of EPCs from the bone marrow.
      • Gill M
      • Dias S
      • Hattori K
      • et al.
      Vascular trauma induces rapid but transient mobilization of VEGFR2(+)AC133(+) endothelial precursor cells.
      EPCs may contribute up to 25% of endothelial cells in newly formed vessels,
      • Murayama T
      • Tepper O
      • Silver M
      • et al.
      Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization in vivo.
      and there is evidence that the biological activity of EPCs and vasculogenesis are impaired in conditions such as diabetes.
      • Tepper O.M
      • Galiano R.D
      • Capla J.M
      • et al.
      Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion and incorporation into vascular structures.
      Figure thumbnail gr1
      Fig. 1Schematic showing the various stages in new vessel formation (including angiogenesis, vasculogenesis and arteriogenesis). Ten individual steps are described in panels A to D showing endothelial cell (EC) proliferation and migration (angiogenesis) followed by extracellular matrix (ECM) remodelling and expansion of vascular smooth muscle (VSM) cells (arteriogenesis).
      A more recently defined term, arteriogenesis, refers to an increase in the calibre of pre-existing arteriolar collateral connections by recruitment of perivascular cells and expansion and remodelling of the extracellular matrix.
      • Ito W.D
      • Arras M
      • Scholz D
      • Winkler B
      • Htun P
      • Schaper W
      Angiogenesis but not collateral growth is associated with ischaemia after femoral artery occlusion.
      Arteriogenesis increases the size and wall thickness of collateral vessels, and shear stress (rather than hypoxia) seems to be the main factor that stimulates arteriogenesis.
      • Schaper W
      • Ito W.D
      Molecular mechanisms of coronary collateral vessel growth.
      Thus, in response to occlusion or stenosis of a major artery the haemodynamic changes in proximal vessels lead to increased blood flow through preformed collateral arterioles. The associated increases in shear stress promote arteriogenesis involving monocyte invasion of the wall of the growing collateral arteriole.
      • Arras M
      • Ito W.D
      • Scholz D
      • et al.
      Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb.
      Migration of circulating monocytes and their differentiation into macrophages within the blood vessel wall is a key element of arteriogenesis, together with recruitment and expansion of smooth muscle cell and matrix components of the vessel wall.
      Thus, the initiation and development of new vessels to provide an effective collateral circulation in occlusive arterial disease involves all three processes of angiogenesis, vasculogenesis and arteriogenesis.

      3. Regulation of Angiogenesis

      Initiation of angiogenesis requires the normally quiescent vascular endothelium to become activated, e.g. by ischaemia or circulating growth factors, which trigger individual cells to break their intercellular adhesions with neighbouring endothelial cells. One ‘leader cell’ then begins to migrate, followed by other cells, in a process of capillary budding and endothelial cell proliferation (Fig. 1). Local release of matrix metalloproteinases (MMPs) and plasminogen degrades the surrounding ECM while the migrating endothelial cells form bands which develop into loops and eventually canalize to allow blood to flow. The new endothelial cells re-establish intercellular connections, and in the later stages of angiogenesis recruitment of smooth muscle cells and fibroblasts creates mature thicker-walled vessels (Fig. 1).
      The key steps in angiogenesis—namely endothelial cell activation, migration, proliferation and reorganisation—are tightly regulated in a complex balance between pro- and anti-angiogenic mechanisms. Over the last 20–30 years, a large number of molecules have been identified which either stimulate or inhibit angiogenesis (Table 1) . The most important pro-angiogenic growth factors are vascular endothelial growth factor (VEGF) (there are four spliced variants of VEGF containing 121, 165, 189 and 206 amino acids),
      • Tischer E
      • Mitchell R
      • Hartman T
      • et al.
      The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing.
      and basic fibroblast growth factor (bFGF), also known as FGF-2 (one of nine members of the FGF family). VEGF is an endothelial cell specific mitogen that is markedly upregulated by hypoxia,
      • Shweiki D
      • Itin A
      • Soffer D
      • Keshet E
      Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.
      and plays an important role in endothelial cell proliferation, differentiation and survival. Platelets are a major source of circulating VEGF,
      • Salgado R
      • Benoy I
      • Bogers J
      • et al.
      Platelets and vascular endothelial growth factor (VEGF): a morphological and functional study.
      which interacts with three tyrosine-kinase receptors (flt-1, flk-1 and flt-4) to promote neovascularization and increased vascular permeability. Production of a soluble form of the flt-1 receptor, s.flt-1, may be important in determining VEGF responses, and there is evidence that s.flt-1 production may be impaired in PAD.
      Table 1Factors that stimulate and inhibit angiogenesis
      StimulateInhibit
      VEGFAngiostatin
      aFGFThrombospondin
      bFGFEndostatin
      PDGFTroponin-I
      TGFα/βTIMPs
      TNFαSuramin
      HGF
      Angiogenin
      PlGF
      Angiopoetin
      Oestrogen
      Nitric oxide
      VEGF, vascular endothelial growth factor; aFGF and bFGF, acid and basic fibroblast growth factor (FGF-1 and FGF-2, respectively); PDGF, platelet-derived growth factor; TGF, transforming growth factor; TNF, tumour necrosis factor; HGF, hepatocyte growth factor; PlGF, placental growth factor; TIMPs, tissue inhibitors of matrix metalloproteinases.
      FGFs, unlike VEGF, are non-secreted growth factors that are released only during cell death or ischaemic cell injury. FGFs are also powerful endothelial cell mitogens, but the FGF family of cytokines is not endothelial cell specific. At least four high-affinity FGF receptors have been cloned and characterised. In vitro studies have shown that FGF and VEGF have different effects on the angiogenic process, e.g. FGF stimulates vascular cells other than endothelial cells. In vivo the inter-play between positive and negative regulators of new vessel formation creates a complex microenvironment that is often called the ‘angiogenic switch’.
      • Hanahan D
      • Folkman J
      Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.
      • Giordano F
      • Johnson R.S
      Angiogenesis: the role of the microenvironment in flipping the switch.
      Cellular interactions are also very important in determining the response of endothelial cells to proliferate and migrate. There is evidence, for example, that leucocytes and platelets exert important effects on endothelial cells and angiogenesis either by direct intercellular contact or via local release of cytokines.
      • Risau W
      Mechanisms of angiogenesis.
      • Pipili-Synetos E
      • Papadimitriou E
      • Maragoudakis M
      Evidence that platelets promote tube formation by endothelial cells on matrigel.
      Clinical and biochemical factors also influence the formation of, and biological response to, different angiogenic growth factors. For example, hypoxia is one of the most potent inducers of angiogenesis,
      • Giordano F
      • Johnson R.S
      Angiogenesis: the role of the microenvironment in flipping the switch.
      principally via up-regulation of VEGF,
      • Shweiki D
      • Itin A
      • Soffer D
      • Keshet E
      Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.
      whereas diabetes and raised levels of cholesterol and lipoprotein (a) are associated with a reduced angiogenic response.
      • Waltenberger J
      Impaired collateral development in diabetes: potential mechanisms and therapeutic implications.
      • Jang J
      • Ho H
      • Kwan H
      • Fajardo L
      • Cooke J
      Angiogenesis is impaired by hypercholesterolaemia.
      • Morishita R
      • Sakaki M
      • Yamamoto K
      • et al.
      Impairment of collateral formation in lipoprotein (a) transgenic mice: therapeutic angiogenesis induced by human hepatocyte growth factor gene.
      Glycation of angiogenic growth factors, e.g. bFGF, reduces their activity,
      • Duraisamy Y
      • Slevin M
      • Smith N
      • et al.
      Effect of glycation on bFGF induced angiogenesis and activation of associated signal transduction pathways in vascular endothelial cells: possible relevance to wound healing in diabetes.
      • Facchiano F
      • Lentini A
      • Fogliano V
      • Mancarella S
      • Rossi C
      • Facchiano A
      Sugar induced modification of FGF-2 reduces its angiogenic activity in vivo.
      while hypercholesterolaemia seems to affect angiogenesis indirectly via reduced nitric oxide (NO) availability.
      • Jang J
      • Ho H
      • Kwan H
      • Fajardo L
      • Cooke J
      Angiogenesis is impaired by hypercholesterolaemia.
      Although ischaemia activates physiological mechanisms leading to collateral vessel formation, there is evidence that in clinical practice the endogenous angiogenic response in patients with PAD is often impaired or insufficient. For example, it has been shown that angiogenesis is reduced in the elderly,
      • Rivard A
      • Fabre J.-E
      • Silver M
      • Chen D
      • Toyoaki M
      • Kearney M
      Age-dependent impairment of angiogenesis.
      and in patients with diabetes
      • Tepper O.M
      • Galiano R.D
      • Capla J.M
      • et al.
      Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion and incorporation into vascular structures.
      • Waltenberger J
      Impaired collateral development in diabetes: potential mechanisms and therapeutic implications.
      • Abaci A
      • Oguzhan A
      • Kahraman S
      • et al.
      Effect of diabetes mellitus on formation of coronary collateral vessels.
      or dyslipidaemia.
      • Jang J
      • Ho H
      • Kwan H
      • Fajardo L
      • Cooke J
      Angiogenesis is impaired by hypercholesterolaemia.
      In addition, many of the commonly prescribed cardiovascular drugs may also impair angiogenesis, e.g. ACE inhibitors,
      • Volpert O
      • Ward W
      • Lingen M
      • Chesler L
      • Solt D
      • Johnson M
      • et al.
      Captopril inhibits angiogenesis and slows the growth of experimental tumours in rats.
      statins
      • Weis M
      • Heeschen C
      • Glassford A
      • Cooke J
      Statins have biphasic effects on angiogenesis.
      and non-steroidal anti-inflammatory drugs (NSAIDs).
      • Jones M
      • Wang H
      • Peskar B
      • Levin E
      • Itani R
      • Sarfeh J
      • et al.
      Inhibition of angiogenesis by NSAIDs: insight into mechanisms and implications for cancer growth and ulcer healing.
      Conversely, physical exercise enhances angiogenesis.
      • Laufs U
      • Werner N
      • Link A
      • Endres M
      • Wassmann S
      • Jurgens K
      • et al.
      Physical training increases endothelial progenitor cells, inhibits neointima formation and enhances angiogenesis.
      Therapeutic angiogenesis aims to overcome any limitations of the natural angiogenic response by increasing substantially the local concentrations of angiogenic growth factor(s) in the lower limb or myocardium, either by administering recombinant protein or the gene that codes for an angiogenic growth factor, or by administering EPCs that will synthesize a cocktail of growth factors in the vicinity of new vessel formation.

      4. Experimental Studies of Therapeutic Angiogenesis in Animal Models of Lower Limb and Myocardial Ischaemia

      There has been considerable research into the pharmacodynamics and pharmacokinetics of different therapeutic interventions to increase levels of angiogenic growth factors in animal models of lower limb and myocardial ischaemia. These studies can broadly be divided into three groups: (1) evaluation of different angiogenic growth factors;
      • Lazarous D.F
      • Scheinowitz M
      • Shou M
      • Lazarous D.F
      • Scheinowitz M
      • Biro S
      • Epstein S.E
      • Unger E.F
      Effect of chronic systemic administration of basic fibroblast growth factor on collateral development in the canine heart.
      • Lazarous D.F
      • Shou M
      • Scheinowitz M
      • et al.
      Comparative effects of basic fibroblast growth factor and vascular endothelial growth factor on coronary collateral development and the arterial response to injury.
      • Banai S
      • Jaklitsch M
      • Shou M
      • Lazarous D
      • Scheinowitz M
      • Biro S
      • et al.
      Angiogenic-induced enhancement of collateral flow to ischaemic myocardium by VEGF in dogs.
      • Lopez J
      • Laham R
      • Stamler A
      • Pearlman J
      • Bunting S
      • Kaplan A
      • et al.
      VEGF administration in chronic myocardial ischaemia in pigs.
      • Takeshita S
      • Zheng L
      • Brogi E
      • Kearney M
      • Pu L
      • Bunting S
      • et al.
      Therapeutic angiogenesis. A single intraarterial bouls of VEGF augments revascularization in a rabbit ischaemic hindlimb model.
      • Baffour R
      • Berman J
      • Garb J
      • Rhee S
      • Kaufman J
      • Friedmann P
      Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant bFGF in a rat model of acute lower limb ischaemia: dose-response effect of bFGF.
      • Unger E
      • Banai S
      • Shou M
      • Lazarous D
      • Jaklitsch M
      • Scheinowitz M
      • et al.
      Basic FGF enhances myocardial collateral flow in a canine model.
      • Yang H
      • Deschenes M
      • Ogilvie R
      • Terjung R
      Basic FGF increases collateral blood flow in rats with femoral artery ligation.
      • Morishita R
      • Nakamura S
      • Hayashi S
      • Taniyama Y
      • Moriguchi A
      • Nagano T
      • et al.
      Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischaemia model as cytokine supplement therapy.
      (2) evaluation of different treatment modalities and different routes of administration, e.g. recombinant protein versus gene transfer;
      • Lazarous D.F
      • Shou M
      • Stiber J.A
      • et al.
      Pharmacodynamics of basic fibroblast growth factor: route of administration determines myocardial and systemic distribution.
      • Witzenbichler B
      • Asahara T
      • Murohara T
      • et al.
      Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischaemia.
      and (3) studies evaluating cellular techniques, including chemotactic methods to attract monocytes to ischaemic tissues,
      • Arras M
      • Ito W.D
      • Scholz D
      • et al.
      Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb.
      local administration of EPCs harvested from the peripheral circulation,
      • Kalka C
      • Masuda H
      • Takahashi T
      • et al.
      Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.
      and even autologous bone marrow transplantation to provide EPCs capable of augmenting vasculogenesis and synthesizing multiple angiogenic growth factors.
      • Fuchs S
      • Baffour R
      • Zhou Y.F
      • et al.
      Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischaemia.

      4.1 Administration of recombinant protein

      Several growth factors administered in the form of recombinant proteins have been shown to augment collateral vessel formation in vivo, e.g. VEGF is effective in models of hindlimb and myocardial ischaemia even after single dose administration.
      • Banai S
      • Jaklitsch M
      • Shou M
      • Lazarous D
      • Scheinowitz M
      • Biro S
      • et al.
      Angiogenic-induced enhancement of collateral flow to ischaemic myocardium by VEGF in dogs.
      • Lopez J
      • Laham R
      • Stamler A
      • Pearlman J
      • Bunting S
      • Kaplan A
      • et al.
      VEGF administration in chronic myocardial ischaemia in pigs.
      • Takeshita S
      • Zheng L
      • Brogi E
      • Kearney M
      • Pu L
      • Bunting S
      • et al.
      Therapeutic angiogenesis. A single intraarterial bouls of VEGF augments revascularization in a rabbit ischaemic hindlimb model.
      Similarly, there is good evidence that recombinant bFGF produces dose-dependent increases in collateral vessel formation, capillary density and blood flow in animal models of intermittent claudication and stable angina.
      • Baffour R
      • Berman J
      • Garb J
      • Rhee S
      • Kaufman J
      • Friedmann P
      Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant bFGF in a rat model of acute lower limb ischaemia: dose-response effect of bFGF.
      • Unger E
      • Banai S
      • Shou M
      • Lazarous D
      • Jaklitsch M
      • Scheinowitz M
      • et al.
      Basic FGF enhances myocardial collateral flow in a canine model.
      • Yang H
      • Deschenes M
      • Ogilvie R
      • Terjung R
      Basic FGF increases collateral blood flow in rats with femoral artery ligation.
      Other recombinant angiogenic growth factors also have therapeutic activity in experimental models of PAD, e.g. hepatocyte growth factor (HGF)
      • Morishita R
      • Nakamura S
      • Hayashi S
      • Taniyama Y
      • Moriguchi A
      • Nagano T
      • et al.
      Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischaemia model as cytokine supplement therapy.
      and placental growth factor.
      • Luttun A
      • Tjwa M
      • Moons L
      • Wu Y
      • Angelillo-Scherrer A
      • Liao F
      • et al.
      Revascularization of ischaemic tissues by PIGF treatment and inhibition of tumour angiogenesis, arthritis and atherosclerosis by anti-Flt1.

      4.2 Gene transfer

      Gene transfer is the introduction of foreign DNA into target cells in order to achieve a localised, sustained therapeutic over-expression of the chosen gene. Several different approaches have been evaluated to transfer an angiogenic growth factor gene into vascular endothelial cells, but the success of any technique depends upon the efficiency with which the transgene is introduced and expressed within the target cell population.
      • Vale P
      • Isner J
      • Rosenfield K
      Therapeutic angiogenesis in critical limb and myocardial ischaemia.
      Naked DNA is poorly taken-up, but different types of DNA vectors have been successfully used to increase the efficiency of gene transfer. In cardiovascular research, the most commonly used vectors are either adenoviruses or plasmids, often formulated with liposomes to facilitate the transfer of DNA across the cell membrane. However, even the best methods of gene transfer still encounter significant problems in achieving high enough rates of transfection to result in clinically significant levels of protein production.
      Adenoviral vectors produce a higher efficiency of gene transfer, but there is a risk of triggering an immune response to the viral DNA. In addition, none of the methods of gene transfer ensure that only the target cells are transfected; introducing foreign DNA into non-target cells may cause adverse effects. Thus, more recently there has been considerable interest in ‘ex vivo gene transfer’—i.e. harvesting cells which are then transfected in vitro before being replaced.
      • Ohara N
      • Koyama H
      • Miyata T
      • Hamada H
      • Miyatake S
      • Akimoto M
      • et al.
      Adenovirus-mediated ex vivo gene transfer of bFGF promotes collateral development in a rabbit model of hind limb ischaemia.
      • Ninomiya M
      • Koyama H
      • Miyata T
      • Hamada H
      • Miyatake S
      • Shigematsu H
      • et al.
      Ex vivo gene transfer of bFGF improves cardiac function and blood flow in a swine chronic myocardial ischaemia model.
      This method increases the transfection efficiency and ensures that foreign DNA is only introduced into target cells.
      Several angiogenic genes have been evaluated in experimental models of lower limb and myocardial ischaemia, e.g. VEGF, bFGF, HGF and hypoxia inducible factor (HIF)-1α. VEGF has been the most intensively studied over the past 10 years, and several studies have shown that gene transfer using naked DNA or adenoviral vectors augments collateral formation and tissue perfusion in models of myocardial and hindlimb ischaemia.
      • Takeshita S
      • Tsurumi Y
      • Couffinahl T
      • Asahara T
      • Bauters C
      • Symes J
      • et al.
      Gene transfer of naked DNA encoding for three isoforms of VEGF stimulates collateral development in vivo.
      • Tsurumi Y
      • Takeshita S
      • Chen D
      • Kearney M
      • Rossow S
      • Passeri J
      • et al.
      Direct intramuscular gene transfer of naked DNA encoding VEGF augments collateral development and tissue perfusion.
      • Mack C
      • Magovern C
      • Budenbender K
      • Patel S
      • Schwarz E
      • Zanzonico P
      • et al.
      Salvage angiogenesis induced by adenovirus-mediated gene transfer VEGF protects against ischaemic vascular occlusion.
      • Mack C
      • Patel S
      • Schwarz E
      • Zanzonico P
      • Hahn R
      • Ilercil A
      • et al.
      Biological bypass with the use of adenovirus-mediated gene transfer of complementary deoxyribonucleic acid for VEGF 121 improves myocardial perfusion and function in the ischaemic porcine heart.
      Unlike VEGF, bFGF lacks a secretory signal sequence and is therefore not actively secreted from cells following gene transfer.
      • Tabata H
      • Silver M
      • Isner J
      Arterial gene transfer of aFGF for therapeutic angiogenesis in vivo: critical role of secretion signal in use of naked DNA.
      Thus, in order to achieve a clinical response the bFGF gene would need to be modified to add a signal sequence prior to transfection. This added difficulty has resulted in less interest in developing gene transfer methods for bFGF compared with VEGF, but limited success has been reported with adenoviral and ex vivo gene transfer of FGFs in animal models of myocardial and hindlimb ischaemia.
      • Ohara N
      • Koyama H
      • Miyata T
      • Hamada H
      • Miyatake S
      • Akimoto M
      • et al.
      Adenovirus-mediated ex vivo gene transfer of bFGF promotes collateral development in a rabbit model of hind limb ischaemia.
      • Ninomiya M
      • Koyama H
      • Miyata T
      • Hamada H
      • Miyatake S
      • Shigematsu H
      • et al.
      Ex vivo gene transfer of bFGF improves cardiac function and blood flow in a swine chronic myocardial ischaemia model.
      • Giordano F
      • Ping P
      • McKirnan M
      • Nozaki S
      • DeMaria A
      • Dillman W
      • et al.
      Intracoronary gene transfer of FGF-5 increases blood flow and contractile function in an ischaemic region of the heart.
      HGF has also shown some potential in gene transfer studies in experimental animals, e.g. transfection of naked DNA augmented collateral vessel growth in a model of hindlimb ischaemia.
      • Taniyama Y
      • Morishita R
      • Hiraoka K
      • Aoki M
      • Nakagami H
      Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat diabetic hind limb ischaemia model.
      HIF-1α is a transcription factor that regulates the expression of several genes encoding angiogenic proteins including VEGF and its receptors. An active form of the transcription factor has been synthesized and transferred effectively in animal studies to induce collateral growth.
      • Vincent K
      • Shyu K.-G
      • Yuxia L
      • Magner M
      • Tio R
      • Jiang C
      • et al.
      Angiogenesis is induced in a rabbit model of hindlimb ischaemia by naked DNA encoding HIF-1 alpha/VP16 hybrid transcription factor.
      • Shyu K.-G
      • Wang M
      • Wang B.-W
      • Chang C
      • Leu J
      • Kuan P
      • et al.
      Intramyocardial injection of naked DNA encoding HIF-1 alpha/VP16 hybrid to enhance angiogenesis in an acute myocardial infarction model in the rat.

      4.3 Delivery of cells that express multiple angiogenic cytokines

      There has always been a concern that angiogenesis may be too complex a process to be stimulated effectively by administration of a single angiogenic cytokine, and therefore a separate line of research has explored ways of increasing cellular recruitment with a view to increasing local production of a cocktail of growth factors. For example, there is evidence that circulating monocytes play a crucial role in arteriogenesis,
      • Arras M
      • Ito W.D
      • Scholz D
      • et al.
      Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb.
      and that differentiation of monocytes into tissue macrophages leads to local secretion of VEGF, nitric oxide and angiogenic cytokines. Thus, in vitro studies have shown that administration of monocyte chemoattractant protein-1 (MCP-1), which increases the recruitment of monocytes to ischaemic tissue, improves collateral flow in a rabbit model of hindlimb ischaemia.
      • Arras M
      • Ito W.D
      • Scholz D
      • et al.
      Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb.
      • Ito W.D
      • Arras M
      • Winkler B
      • et al.
      Monocyte chemoattractant protein-1 increases collateral and peripheral conductance after femoral artery occlusion.
      EPCs derived from bone marrow are present in the peripheral circulation, and are mobilized and incorporated into sites of neovascularization in response to tissue ischaemia.
      • Asahara T
      • Masuda H
      • Takahashi T
      • Kalka C
      • Pastore C
      • Silver M
      • et al.
      Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological neovascularization.
      This has led to the investigation of ‘supply-side angiogenesis’ whereby EPCs are harvested from the bone marrow or peripheral blood, expanded and concentrated ex vivo and then re-administered into the lower limb of the animal.
      • Iba O
      • Matsubara H
      • Nozawa Y
      • Fujiyama S
      • Amano K
      • Mori Y
      • et al.
      Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischaemic limbs.
      • Kawamoto A
      • Tkebuchava T
      • Yamaguchi J
      • Nishimura H
      • Yoon Y
      • Milliken C
      • et al.
      Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischaemia.
      A further development of this technique involves treating the EPCs with adenovirus-containing VEGF or bFGF prior to re-implantation.
      • Iwaguro H
      • Yamaguchi J
      • Kalka C
      • Murasawa S
      • Masuda H
      • Hayashi S
      • et al.
      Endothelial progenitor cell VEGF gene transfer for vascular regeneration.
      The initial results have shown augmented neovascularization and increased perfusion in animal models of myocardial and hindlimb ischaemia.
      • Iwaguro H
      • Yamaguchi J
      • Kalka C
      • Murasawa S
      • Masuda H
      • Hayashi S
      • et al.
      Endothelial progenitor cell VEGF gene transfer for vascular regeneration.
      • Ninomiya M
      • Koyama H
      • Miyata T
      • Hamada H
      • Miyatake S
      • Shigematsu H
      • Taka S
      Ex vivo gene transfer of basic fibroblast growth factor improves cardiac function and blood flow in a swine chronic myocardial ischaemia model.
      Other cellular based angiogenic techniques have evaluated bone marrow derived mononuclear cells and embryonic stem cells.
      • Laham R.J
      • Oettgen P
      Bone marrow transplantation for the heart: fact or fiction.
      • Kamihata H
      • Matsubara H
      • Nishiue T
      • et al.
      Implantation of bone marrow mononuclear cells into ischaemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands and cytokines.

      4.4 Pharmacokinetic and pharmaceutical aspects of therapeutic angiogenesis

      There is uncertainty about the optimum method and frequency of delivery of angiogenic growth factors, whether in the form of recombinant protein or as gene therapy. Ideally, the protein or gene therapy should be easy to administer and produce a sustained, high local concentration of the angiogenic cytokine with low systemic availability. In practice, the i.v. or i.a. routes of administration require massive doses in order to achieve a localised therapeutic effect. This often results in high systemic plasma concentrations and potentially serious side effects.
      • Hariwala M
      • Horowitz J
      • Esakof D
      • Sheriff D
      • Walter D
      • Keyt B
      • et al.
      VEGF improves myocardial blood flow but produces EDRF-mediated hypotension in porcine hearts.
      It has been shown that administration of recombinant bFGF into a peripheral vein is ineffective, whereas local i.a. injection achieves higher local concentrations and a clear angiogenic effect.
      • Sato K
      • Laham R.J
      • Pearlman J.D
      • Novicki D
      • Sellke F.W
      • Simons M
      Efficacy of intracoronary versus intravenous FGF-2 in a pig model of chronic myocardial ischaemia.
      In the case of gene transfer, transfection efficiency is particularly poor when naked DNA is injected into the circulation, probably because of degradation by circulating nucleases. Nevertheless, animal studies have shown a therapeutic effect following i.v. or i.a. injection of gene transfer vectors.
      • Takeshita S
      • Zheng L
      • Brogi E
      • Kearney M
      • Pu L
      • Bunting S
      • et al.
      Therapeutic angiogenesis. A single intraarterial bouls of VEGF augments revascularization in a rabbit ischaemic hindlimb model.
      • Witzenbichler B
      • Asahara T
      • Murohara T
      • Silver M
      • Spyridopoulos I
      • Magner M
      • et al.
      VEGF-C promotes angiogenesis in the setting of tissue ischaemia.
      Intramyocardial injection is more effective in the heart,
      • Kornowski R
      • Leon M.B
      • Fuchs S
      • et al.
      Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy. Results in normal and ischaemic porcine models.
      and gene transfer by intramuscular injection has been successful in increasing collateral growth in animal models of PAD.
      • Mack C
      • Magovern C
      • Budenbender K
      • Patel S
      • Schwarz E
      • Zanzonico P
      • et al.
      Salvage angiogenesis induced by adenovirus-mediated gene transfer VEGF protects against ischaemic vascular occlusion.
      • Vincent K
      • Shyu K.-G
      • Yuxia L
      • Magner M
      • Tio R
      • Jiang C
      • et al.
      Angiogenesis is induced in a rabbit model of hindlimb ischaemia by naked DNA encoding HIF-1 alpha/VP16 hybrid transcription factor.
      • Stark J
      • Baffour R
      • Garb J
      • Kaufman J
      • Berman J
      • Rhee S
      • et al.
      bFGF stimulates angiogenesis in the hindlimb of hyperglycaemic rats.

      5. Clinical Trials of Therapeutic Angiogenesis: Theoretical Risks and Current Information on Safety and Tolerability

      A fairly large number of clinical studies have been reported in the literature, ranging from uncontrolled case reports to larger randomized controlled trials, using several treatment modalities (e.g. recombinant protein, gene transfer or cellular implantation) in patients with PAD (Fontaine stages II and III) and those with endstage myocardial ischaemia. These studies have used a variety of treatments, patient selection criteria and endpoints, as well as different study designs and duration of therapy, therefore meaningful comparisons are difficult and there is very little scope to undertake combined analyses of treatment efficacy. Establishing the safety of novel treatments has been an early priority for clinical studies.
      Pharmacological stimulation of angiogenesis in humans raises a number of theoretical concerns, especially in relation to side effects in non-target tissues, e.g. unwanted neovascularization, tumour growth, haemorrhage from fragile new vessels, and even an adverse effect on atherosclerotic plaques. In addition, gene transfer techniques raise uncertainty about the hazards of introducing foreign DNA which, following intramuscular or intramyocardial injection, may disturb muscle cell growth and turnover.
      • Isner J.M
      • Vale P.R
      • Symes J.F
      • Losordo D.W
      Assessment of risks associated with cardiovascular gene therapy in human subjects.
      The possibility of causing vascular malformations was illustrated in the first-ever report of VEGF gene transfer for PAD; the patient concerned developed three spider angiomas on the treated leg several weeks after DNA administration.
      • Isner J.M
      • Pieczek A
      • Schainfeld R
      • Hakey L
      • Ashasra T
      • Rosenfield K
      Clinical evidence of angiogenesis after arterial gene transfer of phVEGF in patient with ischaemic limb.
      Triggering neovascularization in patients with diabetic retinopathy has been another major concern, particularly since VEGF plays an important role in new vessel formation around the optic disc and in sight-threatening macular oedema.
      • Aiello L
      • Avery R
      • Arrigg P
      • Keyt B
      • Jampel H
      • Shah S
      • et al.
      VEGF in the ocular fluid of patients with diabetic retinopathy and other retinal disorders.
      Most clinical studies of therapeutic angiogenesis have included fundoscopic surveillance and so far no significant adverse effects have been reported in the eye
      • Laham R
      • Chronos N
      • Pike M
      • Leimbach M
      • Udelson J
      • Pearlman J
      • et al.
      Intracoronary FGF-2 in patients with severe ischaemic heart disease: results of a phase I open-label dose escalation study.
      • Shyu K.-G
      • Chang H
      • Wang B.-W
      • Kuan P
      Intramuscular VEGF gene therapy in patients with chronic critical leg ischaemia.
      but several studies have excluded patients with pre-existing diabetic retinopathy (even the common form of background retinopathy). Given that patients with diabetes are an important subgroup that may benefit from therapeutic angiogenesis more safety data are required in this population.
      A number of growth factors are important in tumourigenic angiogenesis, which raises concerns that underlying polyps, tumours and various benign abnormalities might be activated, or develop complications, as a result of systemic exposure to high pharmacological doses of angiogenic cytokines. These concerns are plausible and difficult to exclude, although no serious tumour-related side effects have emerged in clinical trials to date. In fact, in the recent VIVA trial, there were more new diagnoses of malignant disease in the control group.
      • Henry T
      • Annex B
      • McKendall G
      • Azrin M
      • Lopez J
      • Giordano F
      • et al.
      The VIVA Trial: VEGF in ischaemia for vascular angiogenesis.
      Angiogenic growth factors may also stimulate neovascularization within the intima of the arterial wall and at sites of atherosclerotic disease, potentially causing plaque instability and plaque rupture due to intimal neovascularization.
      • Moulton K.S
      Plaque angiogenesis: its functions and regulation.
      The limited results of clinical trials, however, seem to refute this possibility, and indeed, by contrast, suggest that angiogenic factors inhibit neointimal thickening.
      • Laitinen M
      • Hartikainen J
      • Hiltunen M
      • Eranen J
      • Kiviniemi M
      • Manninen H
      • et al.
      Catheter mediated VEGF gene transfer to human coronary arteries after angioplasty.
      Administration of high systemic concentrations of VEGF (also known as vascular permeability factor) has the potential to cause hypotension and oedema. These side effects have been reported in trials of recombinant VEGF, but seem to be relatively mild, transient and reversible.
      • Henry T
      • Annex B
      • McKendall G
      • Azrin M
      • Lopez J
      • Giordano F
      • et al.
      The VIVA Trial: VEGF in ischaemia for vascular angiogenesis.
      • Henry T
      • Rocha-Singh K
      • Isner J
      • Kereiakes D
      • Giordano F
      • Simons M
      • et al.
      Intracoronary administration of recombinant human VEGF to patients with coronary artery disease.
      In the case of recombinant bFGF, the main adverse effects are on the kidney, especially proteinuria.
      • Lazarous D
      • Unger E
      • Epstein S
      • Steine A
      • Arevalo J
      • Chew E
      • et al.
      Basic fibroblast growth factor in patients with intermittent claudication: results of a phase I trial.
      Modest increases in urinary albumin excretion rate have been reported in several studies following single or double doses of bFGF, but one trial of repeated intravenous infusions of bFGF was terminated because of severe proteinuria in five out of 24 patients.
      • Cooper L
      • Hiatt W
      • Creager M
      • Regensteiner J
      • Casscells W
      • Isner J
      • et al.
      Proteinuria in a placebo-controlled study of bFGF for intermittent claudication.
      Notwithstanding the potential hazards of gene transfer,
      • Isner J.M
      • Vale P.R
      • Symes J.F
      • Losordo D.W
      Assessment of risks associated with cardiovascular gene therapy in human subjects.
      one major advantage of this technique is that systemic plasma concentrations of the gene product do not increase,
      • Grines C
      • Watkins M
      • Helmer G
      • Penny W
      • Brinker J
      • Marmur J
      • et al.
      Angiogenic gene therapy (AGENT) trial in patients with stable angina pectoris.
      • Hedman M
      • Hartikainen J
      • Sylvanne M
      • Stjernvall J
      • Hedman A
      • Kivela A
      • et al.
      Safety and feasibility of catheter-based local intracoronary VEGF gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischaemia: Phase II results of the Kuopio Angiogenesis Trial (KAT).
      i.e. the therapeutic effect is very well contained locally. Gene therapy, however, may provoke an unwanted inflammatory response. Several studies have reported transient fevers following the procedure, especially with adenoviral vectors.
      • Hedman M
      • Hartikainen J
      • Sylvanne M
      • Stjernvall J
      • Hedman A
      • Kivela A
      • et al.
      Safety and feasibility of catheter-based local intracoronary VEGF gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischaemia: Phase II results of the Kuopio Angiogenesis Trial (KAT).
      • Makinen K
      • Manninen H
      • Hedman M
      • Matsi P
      • Mussalo H
      • Alhava E
      • et al.
      Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study.
      In one randomized controlled trial, 61% of patients developed adenoviral antibodies which may have important limitations for the feasibility of giving repeated treatments.
      • Makinen K
      • Manninen H
      • Hedman M
      • Matsi P
      • Mussalo H
      • Alhava E
      • et al.
      Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study.
      Most clinical studies to-date have been relatively short (2 weeks to 1 year follow-up), but two recent trials have provided longer-term safety data. Firstly, outcomes up to 3 years after administration of bFGF via perivascular beads at the time of coronary artery bypass grafting showed two late deaths in the highest dose group, one from pancreatic carcinoma and one sudden death of unknown cause.
      • Ruel M
      • Laham R
      • Parker J.A
      • Post M
      • Ware A
      • Simons M
      • et al.
      Long-term effects of surgical angiogenic therapy with FGF-2 protein.
      There were no differences in overall mortality between the actively treated and control groups, and no other longer term serious adverse events.
      • Ruel M
      • Laham R
      • Parker J.A
      • Post M
      • Ware A
      • Simons M
      • et al.
      Long-term effects of surgical angiogenic therapy with FGF-2 protein.
      Secondly, a trial of gene transfer of VEGF using plasmid-liposome and adenoviral vectors for critical limb ischaemia and intermittent claudication has reported safety data up to 2 years. There have been no differences in mortality, and no new tumours detected.
      • Makinen K
      • Manninen H
      • Hedman M
      • Matsi P
      • Mussalo H
      • Alhava E
      • et al.
      Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study.

      6. Clinical Trials of Therapeutic Angiogenesis in Lower Limb Ischaemia: Information About Treatment Efficacy

      Following the first case report of therapeutic angiogenesis in 1996, VEGF gene transfer in a patient with critical limb ischaemia,
      • Isner J.M
      • Pieczek A
      • Schainfeld R
      • Hakey L
      • Ashasra T
      • Rosenfield K
      Clinical evidence of angiogenesis after arterial gene transfer of phVEGF in patient with ischaemic limb.
      there have been numerous clinical studies of different angiogenic agents in both PAD and inoperable myocardial ischaemia. The results have been inconsistent, and several studies were not adequately controlled or powered to make firm conclusions about treatment efficacy. Furthermore, it has been particularly difficult to undertake a combined meta-analysis because the various studies have used different treatment modalities, endpoints and inclusion/exclusion criteria. The placebo effect in cardiovascular interventions should not be underestimated, yet several clinical studies, for obvious practical reasons, were not fully blinded or did not include a properly matched control group. More recently, however, some larger multicentre randomized controlled trials have been published.

      6.1 Clinical trials in patients with ischaemic heart disease: lessons about safety and trial design that are relevant to future prospects in PAD

      There have been several (mostly small and uncontrolled) clinical studies of therapeutic angiogenesis in patients with endstage myocardial ischaemia (Table 2) . The early results using intramyocardial VEGF gene transfer and intra-coronary injection of bFGF protein showed encouraging improvements in anginal frequency and angiographic scores of collateral growth,
      • Laham R
      • Chronos N
      • Pike M
      • Leimbach M
      • Udelson J
      • Pearlman J
      • et al.
      Intracoronary FGF-2 in patients with severe ischaemic heart disease: results of a phase I open-label dose escalation study.
      • Laitinen M
      • Hartikainen J
      • Hiltunen M
      • Eranen J
      • Kiviniemi M
      • Manninen H
      • et al.
      Catheter mediated VEGF gene transfer to human coronary arteries after angioplasty.
      • Henry T
      • Rocha-Singh K
      • Isner J
      • Kereiakes D
      • Giordano F
      • Simons M
      • et al.
      Intracoronary administration of recombinant human VEGF to patients with coronary artery disease.
      • Losordo D
      • Vale P
      • Symes P
      • Dunnington C
      • Esakof D
      • Maysky M
      • et al.
      Gene therapy for myocardial angiogenesis. Initial clinical results with direct myocardial injection of phVEGF165 as a sole therapy for myocardial ischaemia.
      • Laham R
      • Selke F
      • Edelman E
      • Pearlman J
      • Ware A
      • Brown D
      • et al.
      Local perivascular delivery of bFGF in patients undergoing coronary bypass surgery. Results of a phase 1 randomized, double-blind, placebo-controlled trial.
      • Rosengart T
      • Lee L
      • Patel S
      • Sanborn T
      • Parikh M
      • Bergman G
      Angiogenesis gene therapy: phase 1 assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF 121 cDNA to individuals with clinically significant severe coronary artery disease.
      • Losordo D.W
      • Vale P.R
      • Hendel R.C
      • Milliken C.E
      • Fortuin F.D
      • Cumm N
      • Schatz R.A
      • Asahara T
      • Isner J.M
      • Kuntz R.E
      Phase 1/2 placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor-2 gene transfer by catheter delivery in patients with chronic myocardial ischaemia.
      but less impressive results have appeared from larger multicentre randomized controlled trials
      • Henry T
      • Annex B
      • McKendall G
      • Azrin M
      • Lopez J
      • Giordano F
      • et al.
      The VIVA Trial: VEGF in ischaemia for vascular angiogenesis.
      • Grines C
      • Watkins M
      • Helmer G
      • Penny W
      • Brinker J
      • Marmur J
      • et al.
      Angiogenic gene therapy (AGENT) trial in patients with stable angina pectoris.
      • Hedman M
      • Hartikainen J
      • Sylvanne M
      • Stjernvall J
      • Hedman A
      • Kivela A
      • et al.
      Safety and feasibility of catheter-based local intracoronary VEGF gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischaemia: Phase II results of the Kuopio Angiogenesis Trial (KAT).
      • Simons M
      • Annex B
      • Laham R
      • Kleiman N
      • Henry T
      • Dauerman H
      Pharmacological treatment of coronary artery disease with recombinant FGF-2: FIRST study.
      (Table 2). Changes in surrogate endpoints, such as myocardial perfusion imaging or angiography, are much less clinically relevant than differences in anginal frequency or exercise tolerance. Thus, recent larger clinical trials have included the Seattle Angina Questionnaire (SAQ) and other quality of life indices.
      • Henry T
      • Annex B
      • McKendall G
      • Azrin M
      • Lopez J
      • Giordano F
      • et al.
      The VIVA Trial: VEGF in ischaemia for vascular angiogenesis.
      • Simons M
      • Annex B
      • Laham R
      • Kleiman N
      • Henry T
      • Dauerman H
      Pharmacological treatment of coronary artery disease with recombinant FGF-2: FIRST study.
      Table 2Summary of all published clinical studies of therapeutic angiogenesis in patients with myocardial ischaemia
      Ref.TreatmentNo. of SubjectsFollow upOutcomes
      ActiveControlSafetyEndpointsResults
      • Losordo D
      • Vale P
      • Symes P
      • Dunnington C
      • Esakof D
      • Maysky M
      • et al.
      Gene therapy for myocardial angiogenesis. Initial clinical results with direct myocardial injection of phVEGF165 as a sole therapy for myocardial ischaemia.
      Intra-myocardial VEGF gene560 daysNo SAEsAngina frequencyImproved
      Myocardial perfusionImproved (p<0.05)
      AngiographyImproved collateral flow
      • Laham R
      • Selke F
      • Edelman E
      • Pearlman J
      • Ware A
      • Brown D
      • et al.
      Local perivascular delivery of bFGF in patients undergoing coronary bypass surgery. Results of a phase 1 randomized, double-blind, placebo-controlled trial.
      Perivascular beads bFGF protein1683 years2 deaths: 1 in control 1 in activeAngina frequencyTreatment group angina free
      Myocardial perfusionImproved with high dose (p=0.01)
      • Rosengart T
      • Lee L
      • Patel S
      • Sanborn T
      • Parikh M
      • Bergman G
      Angiogenesis gene therapy: phase 1 assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF 121 cDNA to individuals with clinically significant severe coronary artery disease.
      Intra-myocardial VEGF gene212 months3 deathsAngina classImproved class
      AngiographyImproved collateral scores
      Treadmill exerciseImproved in 50%
      • Laitinen M
      • Hartikainen J
      • Hiltunen M
      • Eranen J
      • Kiviniemi M
      • Manninen H
      • et al.
      Catheter mediated VEGF gene transfer to human coronary arteries after angioplasty.
      Intra-coronary VEGF gene1056 monthsNo SAEsAngiographyNo improvement at 6 months
      • Laham R
      • Chronos N
      • Pike M
      • Leimbach M
      • Udelson J
      • Pearlman J
      • et al.
      Intracoronary FGF-2 in patients with severe ischaemic heart disease: results of a phase I open-label dose escalation study.
      Intra-coronary bFGF protein526 monthsMortality 8% hypotension proteinuriaAngina questionnaireImprovement (p<0.001)
      LV ejection fractionSmall improvement
      MR imagingImproved (p<0.001)
      • Henry T
      • Rocha-Singh K
      • Isner J
      • Kereiakes D
      • Giordano F
      • Simons M
      • et al.
      Intracoronary administration of recombinant human VEGF to patients with coronary artery disease.
      Intra-Coronary VEGF protein1560 daysHypotension flushingAngina classImprovement (p=0.002)
      Myocardial perfusionImproved in 50%
      • Losordo D.W
      • Vale P.R
      • Hendel R.C
      • Milliken C.E
      • Fortuin F.D
      • Cumm N
      • Schatz R.A
      • Asahara T
      • Isner J.M
      • Kuntz R.E
      Phase 1/2 placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor-2 gene transfer by catheter delivery in patients with chronic myocardial ischaemia.
      Intra-myocardial VEGF gene12712 weeksNo SAEsAngina classImproved (p=0.04)
      Treadmill exerciseImproved (p=0.02)
      FIRST
      • Simons M
      • Annex B
      • Laham R
      • Kleiman N
      • Henry T
      • Dauerman H
      Pharmacological treatment of coronary artery disease with recombinant FGF-2: FIRST study.
      Intra-coronary bFGF protein25186180 daysNo difference in mortality/SAEsAngina questionnaireImproved at 90 days (p=0.035)
      Myocardial perfusionNo difference
      Treadmill exerciseNo difference
      AGENT
      • Grines C
      • Watkins M
      • Helmer G
      • Penny W
      • Brinker J
      • Marmur J
      • et al.
      Angiogenic gene therapy (AGENT) trial in patients with stable angina pectoris.
      Intra-coronary FGF-4 gene601910 monthsNo difference in mortality/SAEsTreadmill exerciseImproved compared to placebo
      Stress echoNo difference from baseline
      VIVA
      • Henry T
      • Annex B
      • McKendall G
      • Azrin M
      • Lopez J
      • Giordano F
      • et al.
      The VIVA Trial: VEGF in ischaemia for vascular angiogenesis.
      IV+Intra-coronary VEGF protein11563120 daysHypotension flushingAngina questionnaireImprovement at 120 days (p=0.09)
      Myocardial perfusionNo improvement
      Treadmill exerciseImproved at 120 days (p=0.15)
      KAT II
      • Hedman M
      • Hartikainen J
      • Sylvanne M
      • Stjernvall J
      • Hedman A
      • Kivela A
      • et al.
      Safety and feasibility of catheter-based local intracoronary VEGF gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischaemia: Phase II results of the Kuopio Angiogenesis Trial (KAT).
      Intra-coronary VEGF gene65386 monthsNo difference in mortality/SAEsRe-stenosis rateNo difference from placebo
      Myocardial perfusionImprovement at 6 months (p<0.05)
      The three largest muticentre randomized controlled trials, i.e. the FIRST,
      • Simons M
      • Annex B
      • Laham R
      • Kleiman N
      • Henry T
      • Dauerman H
      Pharmacological treatment of coronary artery disease with recombinant FGF-2: FIRST study.
      VIVA
      • Henry T
      • Annex B
      • McKendall G
      • Azrin M
      • Lopez J
      • Giordano F
      • et al.
      The VIVA Trial: VEGF in ischaemia for vascular angiogenesis.
      and KAT II
      • Hedman M
      • Hartikainen J
      • Sylvanne M
      • Stjernvall J
      • Hedman A
      • Kivela A
      • et al.
      Safety and feasibility of catheter-based local intracoronary VEGF gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischaemia: Phase II results of the Kuopio Angiogenesis Trial (KAT).
      studies, have been fairly disappointing. The FIRST trial, for example, evaluated the effects of intracoronary recombinant bFGF in over 330 patients. Although angina symptom scores were significantly improved after 90 days, treadmill exercise tolerance and myocardial perfusion were no different between the two groups.
      • Simons M
      • Annex B
      • Laham R
      • Kleiman N
      • Henry T
      • Dauerman H
      Pharmacological treatment of coronary artery disease with recombinant FGF-2: FIRST study.
      Similarly, the VIVA and KAT II trials used recombinant VEGF and VEGF gene transfer, respectively, and there were only modest, inconsistent improvements in isolated endpoints.
      • Henry T
      • Annex B
      • McKendall G
      • Azrin M
      • Lopez J
      • Giordano F
      • et al.
      The VIVA Trial: VEGF in ischaemia for vascular angiogenesis.
      • Hedman M
      • Hartikainen J
      • Sylvanne M
      • Stjernvall J
      • Hedman A
      • Kivela A
      • et al.
      Safety and feasibility of catheter-based local intracoronary VEGF gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischaemia: Phase II results of the Kuopio Angiogenesis Trial (KAT).

      6.2 Clinical trials in patients with critical limb ischaemia

      The initial studies of therapeutic angiogenesis in PAD were conducted in patients with critical limb ischaemia (Table 3) . These were patients who were either unsuitable for surgical revascularization, or those who had failed other treatment options and were at high risk of distal amputation. Case reports and small uncontrolled studies appeared to show dramatic benefits. For example, VEGF gene transfer was successful in achieving clinical improvement (e.g. resolution of rest pain), increased density of collateral vessels and increased ABPI
      • Isner J.M
      • Vale P.R
      • Symes J.F
      • Losordo D.W
      Assessment of risks associated with cardiovascular gene therapy in human subjects.
      • Shyu K.-G
      • Chang H
      • Wang B.-W
      • Kuan P
      Intramuscular VEGF gene therapy in patients with chronic critical leg ischaemia.
      • Isner J.M
      • Baumgartner I
      • Rauh G
      • Schainfeld D
      • Blair R
      • Manor O
      • et al.
      Treatment of thromboangiitis obliterans (Buerger's Disease) by intramuscular gene transfer of VEGF: preliminary clinical results.
      • Baumgartner I
      • Pieczek A
      • Manor O
      • Blair R
      • Kearney M
      • Walsh K
      • et al.
      Constitutive expression of ph VEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischaemia.
      (Fig. 2) . However, in a larger randomized controlled trial of VEGF gene therapy, 87% of patients showed improved vascularity on digital subtraction angiography but there were no significant differences between the treatment and placebo groups in terms of restenosis after angioplasty, amputation rates, ulcer healing or severity of rest pain.
      • Makinen K
      • Manninen H
      • Hedman M
      • Matsi P
      • Mussalo H
      • Alhava E
      • et al.
      Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study.
      A further uncontrolled study using a plasmid vector for VEGF gene transfer has shown an 83% improvement in rest pain and 75% improvement in ulcer healing, but the number of patients is too small to draw firm conclusions.
      • Shyu K.-G
      • Chang H
      • Wang B.-W
      • Kuan P
      Intramuscular VEGF gene therapy in patients with chronic critical leg ischaemia.
      Table 3Summary of all published clinical studies of therapeutic angiogenesis in patients with PAD
      Ref.TreatmentNo. of subjectsFollow upOutcomes
      ActiveControlSafetyEndpointsResults
      • Isner J.M
      • Pieczek A
      • Schainfeld R
      • Hakey L
      • Ashasra T
      • Rosenfield K
      Clinical evidence of angiogenesis after arterial gene transfer of phVEGF in patient with ischaemic limb.
      Intra-arterial VEGF165 Gene112 weeks3 angiomasAngiographyIncreased collaterals
      • Isner J.M
      • Baumgartner I
      • Rauh G
      • Schainfeld D
      • Blair R
      • Manor O
      • et al.
      Treatment of thromboangiitis obliterans (Buerger's Disease) by intramuscular gene transfer of VEGF: preliminary clinical results.
      Intra-Muscular VEGF165 Gene614 monthsNo SAEsABPIIncreased in 4 limbs
      AngiographyNew collaterals
      • Baumgartner I
      • Pieczek A
      • Manor O
      • Blair R
      • Kearney M
      • Walsh K
      • et al.
      Constitutive expression of ph VEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischaemia.
      Intra-Muscular VEGF165 Gene96 monthsTransient oedemaABPIIncreased (p=0.028)
      AngiographyIncreased collaterals
      SymptomsReduced rest pain (p=0.043)
      • Lazarous D
      • Unger E
      • Epstein S
      • Steine A
      • Arevalo J
      • Chew E
      • et al.
      Basic fibroblast growth factor in patients with intermittent claudication: results of a phase I trial.
      Intra-arterial bFGF Protein1361 yearMild proteinuriaCalf blood flowImproved (p<0.05)
      SymptomsSome improvement
      • Cooper L
      • Hiatt W
      • Creager M
      • Regensteiner J
      • Casscells W
      • Isner J
      • et al.
      Proteinuria in a placebo-controlled study of bFGF for intermittent claudication.
      IV bFGF Protein168Severe proteinuriaNoneStudy stopped prematurely. No positive results at cessation
      • Lederman R.J
      • Mendelsohn F.O
      • Anderson R.D
      • Saucedo J.F
      • Hermiller J.B
      Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC study).
      TRAFFIC
      Intra-arterial bFGF Protein127636 monthsProteinuriaPeak walking timeIncreased at 90 days (p=0.034)
      ABPIIncreased at 90 days (p=0.037)
      • Makinen K
      • Manninen H
      • Hedman M
      • Matsi P
      • Mussalo H
      • Alhava E
      • et al.
      Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study.
      Intra-arterial VEGF Gene35192 yearsNo SAEsAngiographyImproved vascularity (p=0.03)
      ABPINo difference
      SymptomsNo difference
      Re-stenosis rateNo difference
      • Shyu K.-G
      • Chang H
      • Wang B.-W
      • Kuan P
      Intramuscular VEGF gene therapy in patients with chronic critical leg ischaemia.
      Intra-Muscular VEGF165 Gene246 monthsTransient oedemaABPIImproved (p<0.001)
      AngiographyIncreased collaterals (p<0.01)
      SymptomsReduced rest pain and ulcer healing
      • Rajagopalan S
      • Mohler E.R
      • Lederman R.J
      • et al.
      Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease.
      RAVE
      Intra-Muscular VEGF121 Gene337226 weeksOedemaPeak walking timeNo difference
      ABPINo difference
      SymptomsNo difference
      Figure thumbnail gr2
      Fig. 2Ankle-brachial pressure index (ABPI) before and after plasmid-mediated VEGF165 gene transfer in 24 limbs of 21 patients with rest pain and chronic critical limb ischaemia. Doses varying between 400 and 2000 μg of ph VEGF165 were injected intra-muscularly into the affected limbs, and a repeat dose administered 4 weeks later. Results show the change in ABPI following VEGF. Reproduced from an uncontrolled study, with permission.
      There is considerable interest in cellular based therapy for improving lower limb outcomes in critical ischaemia. For example, in the TACT study, a randomized controlled trial, autologous implantation of bone marrow mononuclear cells, including EPCs, into critically ischaemic limbs produced clinical improvements in rest pain in 39 out of 45 patients. In addition, limbs injected with bone marrow mononuclear cells showed a significant increase in ABPI (>0.1) in 31 out of 45 patients, from 0.35 at baseline to 0.47 after 4 weeks (p<0.001).
      • Tateishi-Yuyama E
      • Matsubara H
      • Murohara T
      • et al.
      Therapeutic angiogenesis for patients with critical limb ischaemia using autologous bone marrow cell transplantation.
      Treadmill walking time also improved (1.6–5.0 min, p<0.001), and ischaemic ulcers were healed in 21 out of 28 patients.
      • Tateishi-Yuyama E
      • Matsubara H
      • Murohara T
      • et al.
      Therapeutic angiogenesis for patients with critical limb ischaemia using autologous bone marrow cell transplantation.
      The investigators in the TACT study used peripheral blood mononuclear cells with 500-fold fewer EPCs as the placebo control.

      6.3 Clinical trials in patients with intermittent claudication

      Two randomized, placebo-controlled trials have recently been reported in patients with intermittent claudication.
      • Lederman R.J
      • Mendelsohn F.O
      • Anderson R.D
      • Saucedo J.F
      • Hermiller J.B
      Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC study).
      • Rajagopalan S
      • Mohler E.R
      • Lederman R.J
      • et al.
      Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease.
      Both were phase II ‘proof of concept’ studies, one using intra-arterial recombinant bFGF
      • Lederman R.J
      • Mendelsohn F.O
      • Anderson R.D
      • Saucedo J.F
      • Hermiller J.B
      Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC study).
      and the other using intra-muscular adenoviral gene transfer of VEGF121.
      • Rajagopalan S
      • Mohler E.R
      • Lederman R.J
      • et al.
      Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease.
      The TRAFFIC study was a randomized, double-blind placebo-controlled trial of single or repeat-dose i.a. recombinant bFGF 30 μg/kg.
      • Lederman R.J
      • Mendelsohn F.O
      • Anderson R.D
      • Saucedo J.F
      • Hermiller J.B
      Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC study).
      The formulation of bFGF used in this study was a 146-amino acid, non-glycosylated, monomeric 16.5 kDa protein which is produced in genetically engineered yeast. The dose (30 μg/kg) was the maximum-tolerated dose of bFGF (limited by acute hypotension) in a phase 1 study of intracoronary perfusion.
      • Laham R
      • Chronos N
      • Pike M
      • Leimbach M
      • Udelson J
      • Pearlman J
      • et al.
      Intracoronary FGF-2 in patients with severe ischaemic heart disease: results of a phase I open-label dose escalation study.
      Patients with intermittent claudication and reproducible exercise tolerance on a treadmill received half the dose of bFGF down each femoral artery via a single arterial puncture and crossover catheter. Patients needed two reproducible (within 20%) Gardner treadmill tests (with peak walking time between 1 and 12 min) during a 30-day screening period in order to be eligible for inclusion. Other inclusion criteria included evidence of infra-inguinal obstructive arterial disease (>70% stenosis of femoral, popliteal, or tibial arteries on angiography) and a resting APBI <0.8 on the most affected limb. All patients received appropriate medical management and risk factor modification. Anyone with a history of malignancy within the past 10 years was excluded, as were those with other exercise limiting symptoms, e.g. arthritis or angina. A total of 377 patients were screened for the TRAFFIC study, and 190 were deemed eligible for randomization.
      Patients were randomized to placebo, single-dose bFGF or two doses of bFGF (a second dose 30 days later). Clinical and demographic details were similar in the three groups (e.g. mean ages 65–69 years; 24–38% were current smokers; 33% were diabetic; and half had undergone previous revascularization for PAD). Most patients (85%) had femoropopliteal disease; 30% had isolated femoropopliteal disease and 45–55% had multiple sites of disease in the femoropopliteal region. Two-thirds of patients had evidence of bilateral disease.
      The maximum walking distance at 90 days was the primary endpoint for the TRAFFIC study, and there were results for over 60 patients in each of the three treatment groups. The trial reported a statistically significant improvement in peak walking time, but there was no difference between single and repeated doses of bFGF: for example, compared with baseline patients in the placebo group increased peak walking time by 0.60 min (14%), patients in the single-dose group increased this time by 1.77 min (34%), and patients in the double-dose group increased by 1.54 min (20%) (Fig. 3) . Results of the intention-to-treat analysis (n=190) showed a significant difference between the three groups (p=0.034). Active therapy also produced a small but significant increase in ABPI in the more affected limb (p<0.04).
      • Lederman R.J
      • Mendelsohn F.O
      • Anderson R.D
      • Saucedo J.F
      • Hermiller J.B
      Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC study).
      However, patients with non-compressible vessels (n=10) and those who withdrew from the study early (n=14) or who were revascularized (n=4) were excluded from this analysis.
      Figure thumbnail gr3
      Fig. 3Results from the TRAFFIC study. Improvement in peak walking time (PWT) 90 days after treatment with intra-arterial placebo (n=58), single-dose bFGF (n=62) or double-dose bFGF (n=54) in patients with intermittent claudication. Single dose vs. placebo, p=0.026; double-dose vs. placebo, p=0.45. Reproduced with permission.
      Three subgroups (smoking, diabetes and older age) were prespecified in the TRAFFIC study because of their potential to influence the primary endpoint. Diabetes, age greater than median (68 years), and non-current smoking status were all associated with lower improvement in peak walking time in response to bFGF; however, only smoking status had an independent effect on peak walking time. Current smokers showed a greater increase in peak walking time (1.25, 2.10, and 2.26 min for placebo, single-dose and double-dose, respectively).
      • Lederman R.J
      • Mendelsohn F.O
      • Anderson R.D
      • Saucedo J.F
      • Hermiller J.B
      Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC study).
      One or two doses of bFGF was generally well tolerated in the TRAFFIC study. Transient acute hypotension was uncommon: two patients in the placebo group (3%), four in the single-dose group (6%) and five in the double-dose group (8%). Corresponding frequencies for development of proteinuria were 3% (placebo), 9% (single-dose) and 11% (double-dose). Seven out of nine patients who developed proteinuria also had diabetes. There were two deaths (one in placebo group and one in double-dose group) during the study. There was no evidence of tumourigenesis or adverse effects on the retina with bFGF administration.
      The RAVE trial was also a phase II multicentre, randomized, placebo-controlled, double-blind study to evaluate the safety and efficacy of AdVEGF121, a replication-deficient adenovirus encoding the 121-amino-acid isoform of VEGF.
      • Rajagopalan S
      • Mohler E.R
      • Lederman R.J
      • et al.
      Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease.
      In total 105 patients with unilateral exercise-limiting intermittent claudication were randomized after a run-in phase to establish that they had reproducible exercise performance on a treadmill (peak walking time 1–10 min). Patients were stratified by diabetes status and randomized to low-dose AdVEGF121, high-dose AdVEGF121 or placebo, administered as 20 intramuscular injections to the index leg in a single session. Over 105 patients were entered in the study, which gave >80% power to detect a difference of 1.5 min in peak walking time.
      • Rajagopalan S
      • Mohler E.R
      • Lederman R.J
      • Saucedo J.F
      • Mendelsohn F.O
      • Olin J
      • et al.
      Regional angiogenesis with VEGF in peripheral arterial disease: design of the RAVE trial.
      The results showed no significant difference in the primary endpoint, change in peak walking time after 12 weeks: e.g. mean values of 1.8±3.2 min (placebo) vs. 1.6±1.9 min (low-dose AdVEGF121) vs. 1.5+3.1 min (high-dose AdvEGF121).
      • Rajagopalan S
      • Mohler E.R
      • Lederman R.J
      • et al.
      Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease.
      Secondary endpoints, including ABPI and quality of life measures, were also unchanged after 12 and 26 weeks. The adenoviral therapy was associated with peripheral oedema.
      • Rajagopalan S
      • Mohler E.R
      • Lederman R.J
      • et al.
      Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease.

      7. Possible Explanations for Clinical Trial Results Being Inconsistent and Inconclusive

      The randomized controlled clinical trials in PAD have produced results that are less consistent than those undertaken in animals and less impressive in terms of the absolute treatment effect. This discrepancy raises the possibility that, in the in vivo clinical situation of patients with vascular disease, it is the responsiveness to angiogenic stimuli that is impaired rather than a problem with the availability of angiogenic growth factors. For example, increased (not decreased) levels of VEGF have been reported in PAD,
      • Blann A.D
      • Lip G.Y
      • McCollum C.N
      Influence of the risk factors for atherosclerosis on levels of soluble adhesion molecules and endothelial markers in peripheral vascular disease.
      which could be interpreted as evidence of a potential defect in VEGF responsiveness. Differences in the production of soluble flt-1 may account for inter-subject differences in VEGF effects. In addition, it is possible that angiogenesis is abnormal in some way in patients with PAD. Both of these possibilities might explain why supplementing the availability of angiogenic growth factors does not necessarily augment angiogenesis in patients with arterial disease. The animal models used in various experimental studies often do not have on-going vascular disease and therefore may not mimic the clinical problem of reduced angiogenic responsiveness.

      8. Conclusion

      The formation of new blood vessels, including collaterals, is a complex physiological process that occurs in adults in response to tissue injury or ischaemia. Neovascularization involves angiogenesis, vasculogenesis and arteriogenesis, and there are several pro- and anti-angiogenic cytokines that regulate endothelial cell migration and proliferation. The endogenous angiogenic response seems to be impaired and/or insufficient in patients with PAD or myocardial ischaemia, and therefore therapeutic angiogenesis seeks to augment collateral vessel formation using local administration of recombinant proteins or genes for angiogenic growth factors, or by re-implantation of EPCs harvested from the bone marrow or peripheral circulation.
      VEGF is a secreted, endothelial cell specific mitogen, whereas the family of FGFs, especially bFGF, are not secreted but stimulate non-endothelial cell types in the angiogenic process. Experimental and clinical studies have evaluated the effects of VEGF gene transfer and recombinant bFGF in animals and humans with critical limb ischaemia, intermittent claudication and endstage myocardial ischaemia. Although the early uncontrolled reports were highly encouraging, more recent results from multicentre randomized controlled trials have been far less convincing. In intermittent claudication, intra-arterial recombinant bFGF improved peak walking time at 90 days in the TRAFFIC study
      • Lederman R.J
      • Mendelsohn F.O
      • Anderson R.D
      • Saucedo J.F
      • Hermiller J.B
      Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (TRAFFIC study).
      but VEGF121 gene transfer was ineffective in the RAVE trial.
      • Rajagopalan S
      • Mohler E.R
      • Lederman R.J
      • et al.
      Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease.
      In critical limb ischaemia, autologous bone marrow transplantation was effective in a randomized controlled trial.
      • Tateishi-Yuyama E
      • Matsubara H
      • Murohara T
      • et al.
      Therapeutic angiogenesis for patients with critical limb ischaemia using autologous bone marrow cell transplantation.
      There is still much to learn about the optimum treatment modality, dosing frequency and route of administration, but intra-arterial recombinant protein therapy is closer to being available for routine use than gene therapy. It is becoming clear that trials of single angiogenic growth factors are not achieving the results anticipated from experimental studies, and therefore administration of multiple agents may be necessary to optimize the angiogenic response.
      • Epstein S
      • Fuchs S
      • Zhou Y.F
      • Baffour R
      • Kornowski R
      Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards.
      For example, the combination of VEGF and bFGF has synergistic effects.
      • Asahara T
      • Bauters C
      • Zheng L.P
      • Takeshita S
      • Bunting S
      • Ferrara N
      • Symes J.F
      • Isner J.M
      Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in vivo.
      If regulatory approval is ever granted for these novel (and no doubt expensive) technologies, therapeutic angiogenesis is likely to be reserved for those patients with severe limb-threatening PAD that is not suitable or has failed with conventional revascularization. Whether a one-off intervention at such an advanced stage can achieve sufficient reperfusion, in a short space of time, to avert amputation is uncertain.
      Thus, can biotechnology produce an effective collateral circulation? At present, this seems more remote than it did perhaps 3 years ago when experimental studies were so encouraging. Angiogenesis is clearly complex, and it may be necessary to adopt a therapeutic strategy that has several components to improve angiogenic responsiveness as well as increasing the availability of angiogenic growth factors.

      Acknowledgements

      We are grateful to Samuel Gray for assistance with the artwork.

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